Novel polyketide derivatives and recombinant methods for making same

ABSTRACT

The invention provides novel erythromycin derivatives in which methyl groups on the macrolactone ring have been substituted with —H, -Et, and/or —OH. The invention also provides reagents such as isolated polynucleotides, vectors comprising the polynucleotides and host cells transformed with the vectors for making the novel compounds. Methods for making the compounds utilizing genetic engineering techniques are also disclosed.

[0001] This application is a continuation-in-part of co-pending U.S.application Ser. No. 07/642,734, filed Jan. 17, 1991.

TECHNICAL FIELD

[0002] The present invention relates to novel polynucleotide sequences,proteins encoded therefrom which are involved in the biosynthesis ofpolyketides, methods for directing the biosynthesis of novel polyketidesusing those polynucleotide sequences and novel derivatives producedtherefrom. In particular, the invention relates to the production ofnovel polyketide derivatives through manipulation of the genes encodingpolyketide synthases.

BACKGROUND OF THE INVENTION

[0003] Polyketides are a large class of natural products that includesmany important antibiotic, antifungal, anticancer, antihelminthic, andimmunosuppressant compounds such as erythromycins, tetracyclines,amphotericins, daunorubicins, avermectins, and rapamycins. Theirsynthesis proceeds by an ordered condensation of acyl esters to generatecarbon chains of varying length and substitution pattern that are laterconverted to mature polyketides. This process has long been recognizedas resembling fatty acid biosynthesis, but with important differences.Unlike a fatty acid synthase, a typical polyketide synthase isprogrammed to make many choices during carbon chain assembly: forexample, the choice of “starter” and “extender” units, which are oftenselected from acetate, propionate or butyrate residues in a definedsequence by the polyketide synthase. The choice of using a full cycle ofreduction-dehydration-reduction after some condensation steps, omittingit completely, or using one of two incomplete cycles (reduction alone orreduction followed by dehydration) is additionally programmed, anddetermines the pattern of keto or hydroxyl groups and the degree ofsaturation at different points in the chain. Finally, thestereochemistry for the substituents at many of the carbon atoms isprogrammed by the polyketide synthase.

[0004] Streptomyces and the closely related Saccharopolyspora genera areproducers of a prodigious diversity of polyketide metabolites. Becauseof the commercial significance of these compounds, a great amount ofeffort has been expended in the study of Streptomyces andSaccharopolyspora genetics. Consequently, much is known about theseorganisms and several cloning vectors and techniques exist for theirtransformation.

[0005] Although many polyketides have been identified, there remains theneed to obtain novel polyketide structures with enhanced properties.Current methods of obtaining such molecules include screening of naturalisolates and chemical modification of existing polyketides, both ofwhich are costly and time consuming. Current screening methods are basedon gross properties of the molecules, i.e. antibacterial, antifungalactivity, etc., and both a priori knowledge of the structure of themolecules obtained or predetermination of enhanced properties arevirtually impossible. Chemical modification of preexisting structureshas been successfully employed to obtain novel polyketides, but stillsuffers from practical limitations to the type of compounds obtainable,largely connected to the poor yield of multistep synthesis and availablechemistry to effect modifications. Modifications which are particularlydifficult to achieve are those involving additions or deletions ofcarbon side chains. Accordingly, there exists a considerable need toobtain molecules wherein such changes can be specified and performed ina cost effective manner and with high yield.

[0006] The present invention solves these problems by providing reagents(specifically, polynucleotides, vectors comprising the polynucleotidesand host cells comprising the vectors) and methods to generate novelpolyketides by de novo biosynthesis rather than by chemicalmodification.

SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides compounds of theformula:

[0008] wherein R₁, R₂, R₃, R₄, R₅, and R₆ are independently selectedfrom Q wherein Q is selected from the group consisting of (a) —H, (b)—Me, (c) -Et, and (d) —OH; L₁ and L₂ are independently —H or —OH; L₃ isD-desosamine or —OH, and L₄ is L-mycarose, L-cladinose or —OH with theproviso that when R₁-R₅ are —Me, R₆ is other than —H or —Me. Preferredcompounds of the invention are those in Q is selected from the groupconsisting of (a), (b) and (c) above or (a), (b) and (d) above or (a),(c) and (d) above or (b), (c) and (d) above or (a) and (b) above or (a)and (c) above or (a) and (d) above or (b) and (c) above or (c) and (d)above and L₁, L₂, L₃ and L₄ are its defined above. Other preferredcompounds include those in which R₁, R₂, R₃, R₄, R₅ and R₆ are all —H or-Et or —OH and L₁, L₂, L₃ and L₄ are as defined above. Still otherpreferred compounds include didesnmethyl, tridesmethyl, tetradesmethyl,pentadesmethyl and hexadesmethyl derivatives of the compounds of formulaX and particularly, di- tri-, tetra-, penta- and hexadesmethylderivatives of erythromycins A and B. Other especially preferredcompounds of formula X include 6,10-didesmethyl-6-ethylerythromycin A,10,12-didesmethyl-12-deoxy-12-ethylerythromycin A,10,12-didesmethyl-12-deoxy-10-hydroxyerythromycin A,6,10,12-tridesmethyl-6,12-diethylerythromycin A,6,10,12-tridesmethyl-6-deoxy-6,12-diethylerythromycin A,10-desmethylerythronolide B, 10-desmethyl-6-deoxyerythronolide B,12-desmethylerythronolide B, 12-desmethyl-6-deoxyerythronolide B,12-(desmethyl-12-ethylerythronolide B,6-desmethyl-6-deoxy-6-ethylerythronolide B, 10-desmethlylerythromycin A,10-desmethyl-12-deoxyerythromycin A,10-desmethyl-6,12-dideoxyerythromycin A, 12-desmethylerythromycin A,12-desmethyl- 12-deoxyerythromiycin A,12-desmethyl-6,12-dideoxyerythromycin A,6-desmethyl-6-ethylerythiromycin A, 12-desmethyl-12-ethylerythromycin A,12-desmethyl-12-deoxy-12-ethylerythromycin A,10-desmethyl-10-hydroxyerythromycin A,12-desmethyl-12-epihydroxyerythromycin A, 10,12-didesmethylertthromycinA, 10,12-didesmethyl-12-deoxyerythromycin A,10,12-didesmethyl-6,12-dideoxyerythromycin A, 10-desmethylerythronolideB, 10-desmethyl-6-deoxyerythronolide B, 12-desmethylerythronolide B,12-desmethyl-6-deoxyerythronolide B, 10-desmethylerythromycin A,10-desmethyl-12-deoxyerythromycin A,10-desmethyl-6,12-dideoxyerythromycini A, 12-desmethylerythromycin A,12-desmethyl-12-deoxyerythromycin A,12-desmethyl-6,12-dideoxyerythromycin A, 10,12-didesmethylerythromycinA, 10,12-didesmethyl-12-deoxyerythromycin A, and10,12-didesmeythyl-6,12-dideoxyerythromycin A. Most preferred compoundsinclude 10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A,and 12-desmethyl-12-deoxyerythromycin A.

[0009] In another aspect, the present invention provides an isolatedpolynucleotide sequence or fragment thereof which encodes anenzymatically active acyltransferase domain from a PKS selected fromStreptomyces hygroscopicus, Streptomyces venezuelae, and Streptomycescaelestis. Preferably, the polynucleotide sequence is SEQ ID NO:1, SEQID NO:2, SEQ ID NO:29 or SEQ ID NO:30. In another preferred embodiment,the polynucleotide sequence encodes an acyltransferase domain selectedfrom the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33and SEQ ID NO:34.

[0010] The present invention also provides a vector comprising apolynucleotide sequence or fragment thereof which encodes which encodesan enzymatically active acyltransferase domain from Streptomyces.Preferably, the polynucleotide sequence is selected from those describedabove and the Streptomyces is Streptomyces hygroscopicus, Streptomycesvenezuelae, or Streptomyces caelestis. A particularly preferred vectoris pCS5. Other vectors of the invention include pUC18/LigAT2,pEryAT1/LigAT2, pEryAT2/LigAT2, pUC18/venAT, pEryAT1/venAT,pUC19/rapAT14, pEryAT1/rapAT14, pEryAT2/rapAT14, pUC/5′-flank/ethAT,pUC/ethAT/C-6, pEAT4, pUC18/NidAT6, and pEryAT2/NidAT6.

[0011] In another aspect, the invention provides host cells transformedwith a vector as described above. The host cell may be a bacterial celland preferably is selected from the group consisting of E. coli andBacillus species. Alternatively, the host cell is a polyketide-producingmicroorganism. A preferred polyketide-producing host cell is selectedfrom the group consisting of Saccharopolyspora species, Nocardiaspecies, Micromonospora species, Arthrobacter species, Streptomycesspecies, Actinomadura species, and Dactylosporangium species. An evenmore preferred polyketide-producing host cell is selected from the groupconsisting of Saccharopolyspora hirsuta, Micromonospora rosaria,Micromonospora megalomicea, Streptomyces antibioticus, Streptomycesmycarofaciens, Streptomyces avermitilis, Streptomyces hygroscopicus,Streptomyces caelestis, Streptomyces tsukubaensis, Streptomyces fradiae,Streptomyces platensis, Streptomyces violaceoniger, Streptomycesambofaciens, Streptomyces griseoplanus, and Streptomyces venezuelae. Ofthese host cells, Streptomyces caelestis are most preferred.

[0012] The invention also provides a method for altering the substratespecificity of a polyketide synthase in a first polyketide-producingmicroorganism comprising the steps of

[0013] (a) isolating a first and second genomic DNA segment, eachcomprising a polyketide synthase wherein the first genomic DNA segmentis from the first polyketide-producing microorganism and the secondgenomic DNA segment is from the first polyketide-producing microorganismor a second polyketide-producing microorganism;

[0014] (b) identifying one or more discrete fragments of the firstgenomic DNA segment, each of which encodes an acyltransferase domain;

[0015] (c) identifying one or more discrete fragments of the secondgenomic DNA segment, each of which encodes a related domain to theacyltransferase domain of the first genomic DNA segment; and

[0016] (d) transforming a cell of the first polyketide-producingmicroorganism with one or more of the fragments from step (c) underconditions suitable for the occurrence of a homologous recombinationevent, leading to the replacement of one or more of the fragments fromthe first genomic DNA segment with one or more of the fragments fromstep (c). In one embodiment, the first polyketide-producingmicroorganism is Saccharopolyspora erythraea and the secondpolyketide-producing microorganism is Steptomyces. PreferredStreptomyces are selected from the group consisting of Streptomycesantibioticus, Steptomyces mycarofaciens, Streptomyces avermitilis,Steptomyces hygroscopicus, Streptomyces caelestis, Streptomycestsukubaensis, Streptomyces fradiae, Streptomyces platensis, Streptomycesviolaceoniger, Streptomyces ambofaciens, and Streptomyces venezuelae.Even more preferred Streptomyces are Streptomyces caelestis,Streptomyces hygroscopicus, or Streptomyces venezuelae. In a secondembodiment, the first polyketide-producing microorganism is aStreptomyces as described above and the second polyketide-producingmicroorganism is Saccharopolyspora erthraea. Also in a preferredembodiment, the related domain is selected from the group consisting ofSEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be more readily appreciated inconnection with the accompanying drawings.

[0018]FIG. 1 is a proposed metabolic pathway for the biosynthesis oferythromycin A in Sac. erythraea.

[0019]FIG. 2 is a schematic representation of the erythromycin PKS.

[0020]FIG. 3 is a Growtree analysis of AT domains from Streptomyceshygroscopicus (S. hygroscopicus; LigAT2 and rapAT1-14), Streptomycesvenezuelae (S. venezuelae; venAT) and Saccharopolyspora erythra (Sac.erythraea; eryAT 1-6).

[0021]FIG. 4a is a schematic representation of gene replacements ofEryAT1 with LigAT2 or venAT and EryAT2 with LigAT2 in Sac. erythraea.

[0022]FIG. 4b is a schematic representation of gene replacements ofEryAT4 with an ethyl AT (NidAT5) in Sac. erythraea.

[0023]FIG. 5 is a diagrammatic representation of gene replacement byhomologous recombination.

[0024]FIG. 6 is a schematic representation of the genetic organizationof the Ligase-PKS cluster from S. hygroscopicus ATCC 29253.

[0025]FIG. 7 represents the nucleotide sequence (SEQ ID NO:1, topstrand) and corresponding amino acid sequence (SEQ ID NO:3, bottomstrand) of LigAT, the malonyl AT domain from module 2 of the Ligase-PKScluster of S. hygroscopicus ATCC 29253.

[0026]FIG. 8 is a diagrammatic representation of the strategy to clonethe LigAT2 domain.

[0027]FIG. 9 is a flow diagram depicting the cloning of the EryAT1flanking regions in plasmid pCS5.

[0028]FIG. 10 is a flow diagram depicting construction ofpEryAT1/LigAT2.

[0029]FIG. 11 is a computer generated PhosphorImage of a Southernanalysis of chromosomal DNA from Sac. erythraea ER720EryAT1/LigAT2resolvants cut with SphI and probed with an approximately 3 kbEcoR1/HindIII fragment from pEryAT1/LigAT2. As shown in lanes 3, 4 and 7the probe hybridized with fragments of 3.5 and 1.6 kb, indicating thatLigAT2 had replaced EryAT1 in the chromosomes of these resolvants (clone#10, #11 and #24 respectively). Lanes 5 and 6: chromosomal DNA from Sac.erythraea ER720 resolvants to wild-type (wt); lanes 1 and 9: molecularweight markers (1 kb ladder).

[0030]FIG. 12 is a computer reproduction of a TLC plate on which theproducts produced by Sac. erythraea ER720 EryAT1/LigAT2 were run. Lanes1 and 7: erythromycin A standard (5 μg); lanes 2 and 6: compoundsproduced by wild-type Sac. erythraea ER720; lanes 3, 4 and 5: compoundsproduced by Sac. erythraea ER720 EryAT1/LigAT2 resolvants tomutant-type, clones #10, #11 and #24 respectively.

[0031]FIG. 13 is a flow diagram depicting the cloning of the EryAT2flanking regions in plasmid pCS5.

[0032]FIG. 14 is a flow diagram depicting construction ofpEryAT2/LigAT2.

[0033]FIG. 15 is a computer generated PhosphorImage of a Southernanalysis of chromosomal DNA from Sac. erythraea ER720EryAT2/LigAT2, cutwith SphI and probed with an approximately 1 kb LigAT2 sequence. As seenin lane 3, an approximately 900 base pair fragment hybridized with theprobe, indicating that LigAT2 had replaced EryAT2 in this resolvant.Lane 2: chromosomal DNA from wild-type (wt) Sac. erythraea a ER720;lanes 1 and 5: molecular weight markers (1 kb ladder).

[0034]FIG. 16 is a computer reproduction of a TLC plate on which theproducts produced by Sac. erythraea ER720EryAT2/LigAT2 were run. Lanes 1and 6: erythromycin B standard (5 μg); lanes 2 and 5: erythromycin Astandard (5 μg); lane 4: compounds produced by wild-type Sac. erythraeaER720; lane 3: compounds produced by Sac. erythraea ER720 EryAT2/LigAT2resolvant, clone #2-4.

[0035]FIG. 17 is a computer reproduction of Xerox image of abioautography plate of products made by Sac. erythraeaER720EryAT2/LigAT2 against S. aureus. Lanes 1 and 7: erythromycin Bstandard (1 μg); lanes 2 and 6: erythromycin A standard; lane 3:compounds produced by wild-type Sac. erythraea ER720; lane 4: extractfrom an 0.1 mL culture of Sar. etythraea ER720 EryAT2/LigAT2 resolvantclone #2-4; lane 5: extract from an 0.5 mL culture of Sac. erythraeaER720 EryAT2/LigAT2 resolvant clone #2-4.

[0036]FIG. 18 represents the nucleotide sequence (SEQ ID NO:2, topstrand) and corresponding amino acid sequence (SEQ ID NO:32, bottomstrand) of venAT, the malonate AT domain from the PKS cluster(hereinafter designated pven4) from S. venezuelae ATCC 15439.

[0037]FIG. 19 is a diagrammatic representation of the strategy to clonethe venAT domain.

[0038]FIG. 20 is a flow diagram depicting construction of pEryAT1/venAT.

[0039]FIG. 21 is a computer generate PhosphorImage of a Southernanalysis of chromosomal DNA from Sac. erythraea ER720 EryAT1/venATresolvants, cut with PvuII and probed with a venAT sequence. As seen inlanes 4 and 5, the probe hybridized with fragments of 4.2 and 2.4 kb,indicating that venAT had replaced EryAT1 in these resolvants. Lane 1:molecular weight markers (1 kb ladder); lane 2: chromosomal DNA from awild-type Sac. erythraea ER720; lane 3: chromosomal DNA from a Sac.erythraea ER720 EryAT1/venAT integrant; lane 4: chromosomal DNA fromSac. erythraea ER720 EryAT1/venAT resolvant clone #C.1; lane 5:chromosomal DNA from Sac. erythrea ER720 EryAT1/venAT resolvant clone#C.4.

[0040]FIG. 22 is a computer reproduction of a TLC plate on which theproducts produced by Sac. erythraea ER720 EryAT1/venAT were run. Lane 1:erythromycin A standard (EryA; 5 μg) and 3-α-mycarosylerythronolide B(MEB; 10 μg); lane 2: compounds produced by Sac. erythraea ER720EryAT1/venAT resolvant clone #C.4.

[0041]FIG. 23 is a diagrammatic representation of the strategy to clonethe rapAT14 domain.

[0042]FIG. 24 is a flow diagram depicting construction ofpEryAT1/rapAT14.

[0043]FIG. 25 is a computer generated PhosphorImage of a Southernanalysis of chromosomal DNA from an Sac. erythraea ER720EryAT1/rapAT14resolvant, cut with StyI and probed with an EcoRI-HindIII fragment frompCS5AT1-flank. As shown in lane 2 the probe hybridized with a 1.6 kbfragment indicating that rapAT14 had replaced EryAT1 in the chromosomeof this resolvant. Lane 1: molecular weight markers (1 kb ladder); lane2: chromosomal DNA from Sac. erythraea ER720 EryAT1/rapAT14 resolvantclone #4-A(1); lane 3: chromosomal DNA from wild-type Sac. erythraeaER720.

[0044]FIG. 26 is a computer reproduction of a TLC plate on which theproducts produced by Sac. erythraea ER720 EryAT1/rapAT14 were run lane1: erythromycin A standard (5 μg); lane 2: compounds produced by Sac.erythraea ER720 EryAT1/rapAT14 resolvant.

[0045]FIG. 27 is a flow diagram depicting construction ofpEryAT2/rapAT14.

[0046]FIG. 28 is a computer generated PhosphorImage of a Southernanalysis of chromosomal DNA from Sac. erythraea ER720 EryAT2/rapAT14resolvants, cut with BspHI and probed with a fragment of 5′-flankingregion of eryAT2. As shown in lanes 5, 6 and 7, the probe hybridizedwith a 4.3 kb fragment, indicating that rapAT14 had replaced EryAT2 inthe chromosomes of these resolvants. Lane 1: molecular weight markers (1kb ladder); lane 2: chromosomal DNA from wild-type Sac. erythraea ER720;lane 3: chromosomal DNA from Sac. erythraea ER720 resolvant towild-type, clone #1.1; lane 4: chromosomal DNA from a Sac. erythraeaER720 EryAT2/rapAT14 integrant; lanes 5-7: chromosomal DNA from Sac.erythraea ER720 EryAT2/rapAT14 resolvant clones #1.2, #1.3 and #1.4respectively.

[0047]FIG. 29 is a computer reproduction of a TLC plate on which theproducts produced by Sac. erythraea ER720 EryAT2/rapAT14 were run. Lane1: erythromycin A and erythronolide B (EryA and EB, respectively; 5 μgeach); lane 2: compounds produced by wild-type Sac. erythraea ER720;lanes 3-5: compounds produced by Sac. erythraea ER720 EryAT2/rapAT14resolvant clones #1.2, #1.3 and #1.4 respectively.

[0048]FIG. 30 is a computer reproduction of a bioassay of compounds madeby Sac. erythraea ER720 EryAT2/rapAT14 resolvant clones #1.2, #1.3 and#1.4 and resolvant to wild-type clone #1.1.

[0049]FIG. 31 is a computer generated PhosphorImage of a Southernanalysis of a cosmid DNA library constructed from Streptomyces caelestisNRRL-2821 chromosonal DNA. Lanes 1-19: DNA prepared from 19 clones,digested with SstI and probed with a Streptomyces caelestis NRRL-2821PKS specific probe.

[0050]FIG. 32 is a schematic representation of the genetic organizationof the PKS cluster from Streptomyces caelestis NRRL-2821.

[0051]FIG. 33 is a diagram of the structure of the macrolide ring ofniddamycin.

[0052]FIG. 34 represents the nucleotide sequence (SEQ ID NO:29, topstrand) and corresponding amino acid sequence (SEQ ID NO:33, bottomstrand) of NidAT5, the ethyl AT domain from module 5 of the PKS clusterof Streptomyces caelestis NRRL-2821.

[0053]FIG. 35 is a flow diagram depicting the construction ofpUC/ethAT/C-6.

[0054]FIG. 36 is a diagram showing the nucleotide changes made to createan AvrIl site at the 5′ end of NidAT5.

[0055]FIG. 37 is a diagram of the replacement plasmid pEAT4.

[0056]FIG. 38 is a computer generated PhoshorImage of a Southernanalysis of chromosonal DNA from Sac. erythraea ER720 EAT4 resolvantsdigested with MluI and probed with a 900 bp DNA fragment spanning aKS/AT domain in Streptomyces caelestis NRRL-2821. Lane assignments areas follows: 1) wild type ER720; 2-7) resolvant clones. The resolvantswith the NidAT5 domain in place of EryAT4 produced a stronglyhybridizing 1.8 fragment (lanes 4, 5, and 7) which is missing in cloneswhich resolved back to wild type (lanes 2, 3, and 6).

[0057]FIG. 39 is a computer reproduction of a TLC plate showing theproducts made by Sac. erythraea EAT4-46 after growing in SCM or SCM+50mM butyric acid.

[0058]FIG. 40 is a computer generated PhosphorImage of a Southernanalysis of clones from a cosmid DNA library constructed fromStreptomyces caelestis NRRL-2821 chromosomal DNA. Clones were digestedwith SmaI and probed with a 900 bp DNA fragment spanning a KS/AT domainin Streptomyces caelestis NRRL-2821.

[0059]FIG. 41 represents the nucleotide sequence (SEQ ID NO:30, topstrand) and corresponding aminio acid sequence (SEQ ID NO:34, bottomstrand) of NidAT6, the AT domain in module 6 of the niddamycin PKScluster.

[0060]FIG. 42 is a diagrammatic representation of the strategy to clonethe NidAT6 domain.

[0061]FIG. 43 is a flow diagram depicting construction ofpEryAT2/NidAT6.

DETAILED DESCRIPTION OF THE INVENTION

[0062] I. Definitions:

[0063] For the purposes of the present invention as disclosed andclaimed herein, the following terms are defined:

[0064] The term “polyketide” as used herein refers to a large anddiverse class of natural products including but not limited toantibiotic, anticancer, antihelminthic, antifungal, pigment, andimmunosuppressant compounds. Antibiotics include but are not limited toanthracyclines, tetracyclines, polyethers, polyenes, ansamycins, andmacrolides of various types such as avermectins, erythromycins, andniddamycins. The term polyketide is also intended to refer to compoundsof this class that can be used as intermediates in chemical syntheses.For example, erythromycin A is a polyketide that is isolated and used inthe synthesis of the antibiotic clarithromycin. Polyketides used asintermediates do not themselves necessarily have any biological ortherapeutic activity.

[0065] The term “polyketide-producing microorganism” as used hereinincludes but is not limited to bacteria from the order Actinomycetales,Myxococcales or other Eubacteriales that can produce a polyketide.Examples of actinomycetes and myxobacteria that produce polyketidesinclude but are not limited to Saccharopolyspora erythraea,Saccharopolyspora hirsuta, Micromonospora rosaria, Micromonosporamegalomicea, Sorangium cellulosum, Streptomyces antibioticus,Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyceshygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis,Streptomyces fradiae, Streptomyces platensis, Streptomycesviolaceoniger, Streptomyces ambolfaciens, Streptomyces venezuelae andVarious other Streptomyces, Actinomodura, Dactylosporangium andAmycolotopsis strains that produce polyketides. Yeast and fungi thatproduce polyketides are also considered “polyketide-producingmicroorganisms”. Examples of fungi that produce polyketides include butare not limited to members of the genus Aspergillus.

[0066] The term “polyketide synthase” (PKS) as used herein refers to acomplex of enzyme activities responsible for the biosynthesis ofpolyketides. The enzymatic activities contained within a PKS include butare not limited to β-ketoreductase (KR), dehydratase (DH),enoylreductase (ER), β-ketoacyl ACP synthase (KS), acyl carrier protein(ACP), acyltransferase (AT) and thioesterase (TE). The polypeptidefragment responsible for each enzymatic activity is referred to as a“domain”. A “module” refers to a group or set of domains which carry outone condensation step in the process of polyketide formation and may ormay not include domains which effect processing of the β-carbonyl groupin the growing polyketide.

[0067] The term “Type I PKS” as used herein refers to a PKS which is alarge multifunctional protein and is exemplified by DEBS (see below).The term “Type II PKS” refers to a PKS having several separate, largelymonofunctional enzymnes, and is exemplified by the PKSs responsible forthe biosynthesis of actinorhodin and tetracenomycin (C. R. Hutchinsonand I. Fujii, Annu. Rev. Microbiol. 49:201-238 (1995)).

[0068] The term “cognate domains” as used herein refers to the membersof a specific set of domains which constitute a naturally occurringsingle module.

[0069] The term “related domain” or “heterologous domain” as used hereinrefers to a, PKS domain which is functionally similar to a second PKSdomain. By “functionally similar” it is meant that each domain catalyzesa particular type of reaction but acts upon a different substrate. Forexample, the AT domain of module 1 of Sac. erythraea (eryAT1) and the ATdomain of module 14 of S. hygroscopicus (rapAT14) both catalyze thetransfer of an extender unit to a corresponding ACP domain. In the caseof Sac. erythraea, however, eryAT1 utilizes methylmalonyl CoA as asubstrate whereas in S. hygroscopicus, rapAT14 utilizes malonyl CoA.Thus, eryAT1 and rapAT14 are considered to be “related” or“heterologous” domains.

[0070] The term “condensation” as used herein refers to the addition ofan extender unit to the nascent polyketide chain and requires the actionof KS, AT and ACP domains of the PKS.

[0071] The term “starter” as used herein refers to a coenzyme Athioester of a carboxylic acid which is used by a polyketide synthase asthe first building block of the polyketide.

[0072] The term “extender” as used herein refers to a coenzyme Athioester of a dicarboxylic acid that is incorporated into a polyketideby a polyketide synthase at positions other than the first position.

[0073] The tern “DEBS” as used herein refers to the enzyme6-deoxyerythronolide B synthase, the PKS that builds thepolyketide-derived macrolactone 6-deoxyerythronolide B (6-DEB).

[0074] The term “eryA” as used herein refers to the genes which encodethe DEBS.

[0075] The term “homologous recombination” as used herein refers tocrossing over between DNA strands containing identical sequences.

[0076] The term “isolated” as used herein means that the material isremoved from its original environment (e.g. the natural environmentwhere the material is naturally occurring). For example, a naturallyoccurring polynucleotide or polypeptide present in a living animal isnot isolated, but the same polynucleotide or polypeptide, which isseparated from some or all of the coexisting materials in the naturalsystem, is isolated. Such polynucleotides could be part of a vectorand/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that the vector or composition is,not part of the natural environment.

[0077] The term “restriction fragment” as used herein refers to anylinear DNA generated by the action of one or more restriction enzymes.

[0078] The term “transformation” as used herein refers to theintroduction of DNA into a recipient microorganism, irrespective of themethod used for the insertion into the microorganism.

[0079] The term “replicon” as used herein means any genetic element,such as a plasmid, chromosome or virus, that behaves as an autonomousunit of polynucleotide replication within a cell. A “vector” is areplicon in which another polynucleotide fragment is attached, such asto bring about the replication and/or expression of the attachedfragment.

[0080] The terms “recombinant polynucleotide” or “recombinantpolypeptide” as used herein means at least a polynucleotide orpolypeptide which by virtue of its origin or manipulation is notassociated with all or a portion of the polynucleotide or polypeptidewith which it is associated in nature and/or is linked to apolynucleotide or polypeptide other than that to which it is linked innature.

[0081] The term “host cell” as used herein, refers to both prokaryoticand eukaryotic cells which are used as recipients of the recombinantpolynucleotides and vectors provided herein.

[0082] The term “open reading frame” or “ORF” as used herein refers to aregion of a polynucleotide sequence which encodes a polypeptide; thisregion may represent a portion of a coding sequence or a total codingsequence.

[0083] II. The Invention

[0084] In its broadest sense, the present invention entails novelpolyketides with therapeutic activity (e.g. antimicrobial, anticancer,antifungal, immunosuppressant and/or antihelminthic activity) andimmediate compounds of such polyketides. The invention also provides amethod for producing novel polyketides in vivo by selectively alteringthe genetic information of an organism that naturally produces apolyketide. The present invention further provides isolated and purifiedpolynucleotides that encode PKS domains (i.e. polypeptides frompolyketide-producing microorganisms, fragments thereof, vectorscontaining those polynucleotides, and host cells transformed with thosevectors. These polynucleotides, fragments thereof, and vectorscomprising the polynucleotides can be used as reagents in the abovedescribed method. Portions of the polynucleotide sequences disclosedherein are also useful as primers for the amplification of DNA or asprobes to identify related domains from other polyketide-producingmicroorganisms.

[0085] III. Polynucleotides

[0086] The present invention provides isolated and purifiedpolynucleotides that encode PKS domains (i.e. polypeptides) andfragments thereof which are involved in the production of polyketides.Polynucleotides included within the scope of the invention may be in theform of RNA, DNA, cDNA, genomic DNA and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single-stranded may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence which encodes a polypeptide may be identical to a codingsequence provided herein or may be a different coding sequence which, asa result of the redundancy or degeneracy of the genetic code, encodesthe same polypeptide as the DNA provided herein.

[0087] Polynucleotides may include only the coding sequence for aparticular polypeptide or for a polypeptide which is functionallyequivalent to the polypeptide sequences provided herein. Additionally,the invention includes variant polynucleotides containing modificationssuch as polynucleotide deletions, substitutions or additions; and anypolypeptide modification resulting from the variant polynucleotidesequence. A polynucleotide of the present invention also may have acoding sequence which is a naturally occurring allelic variant of thecoding sequence provided herein.

[0088] Probes and primers constructed according to the polynucleotidesequences provided herein are also contemplated as within the scope ofthe present invention and can be used in various methods to providevarious types of analysis. For example, primer sequences may be designedaccording to polynucleotide sequences which encode particular domainsand then used to amplify polynucleotide sequences of the same or otherrelated domains using well-known amplification techniques such as thepolymerase chain reaction (PCR) and the ligase chain reaction (LCR).(PCR has been disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, andLCR, in EP-A-320 308 to K. Backman published Jun. 16, 1989 and EP-A-439182 to K. Backman et al., published Jul. 31, 1991, all of which areincorporated herein by reference). Generation of primers for use inother amplification techniques or in variations of these amplificationtechniques, (such as nested PCR) is also contemplated within the scopeof the invention and is considered within the knowledge of the routinepractitioner.

[0089] Probes and primers may be designed from conserved nucleotideregions of a polynucleotide of interest or from non-concernednucleotidle regions of a polynucleotide of interest. Generally, nucleicacid probes are developed from non-conserved or unique regions whenmaximum specificity is desired, and nucleic acid probes are developedfrom conserved regions when assaying for nucleotide regions of relatedmembers of a multigene family or in related species. Probes can also belabeled with radioisotopes or other detection labels for screening ofrecombinant libraries.

[0090] Various methods for synthesizing primers and probes arewell-known in the art as are methods for attaching labels to primers orprobes. For example, it is a matter of routine to synthesize desirednucleic acid primers or probes using conventional nucleotidephosphoramidite chemistry and instruments available from AppliedBiosystems, Inc., (Foster City, Calif.), Dupont (Wilmington, Del.), orMilligen (Bedford, Mass.). Many methods have been described for labelingoligonucleotides such as the primers or probes of the present invention.Commercially available probe labeling kits include those from AmershamLife Science (Arlington Heights, Ill.), Promega (Madison, Wis.), EnzoBiochemical (New York, N.Y.) and Clontech (Palo Alto, Calif.).

[0091] IV. Vectors and Host Cells

[0092] The present invention provides vectors which includepolynucleotides of the present invention and host cells which aregenetically engineered with vectors of the present invention.

[0093] a. Vectors and Expression Systems

[0094] The present invention includes recombinant constructs comprisingone or more of the sequences as broadly described above. The constructscomprise a vector, such as a plasmid or viral vector, into which asequence of the invention has been inserted, in a forward or reverseorientation. Such vectors include chromosomal, nonchromosonal andsynthetic DNA sequences from prokaryotic or eukaryotic sources. Largenumbers of suitable plasmids and vectors are known to those of skill inthe art, and are commercially available. Vectors which are particularlyuseful for cloning and expression in intermediate hosts include but arenot limited to: (a) Bacterial: pBR322 (ATCC37017); pGEM (Promega Biotec,Madison, Wis.), pUC, pSPORT1 and ProEX1 (Life Technologies,Gaithersburg, Md.); pQE70, pQE60, pQE-9 (Qiagen); pBs, phagescript,psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene®, La Jolla, Calif.); pTrc99A, pKK223-3, pKK233-3, pDR540,pRIT5, and pGEX4T (Pharmacia®, Piscataway, N.J.); and (b) Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene®); pSVK3, pBPV, pMSG, pSVL(Pharmacia®); pcDNA3.1 (Invitrogen, Carlsbad, Calif.). Other appropriatecloning and expression vectors for use with prokaryotic and eukaryotichosts are described by Maniatis et al., Molecular Cloning: A LaboratoryManual, Second Edition, (Cold Spring Harbor Press, N.Y., 1982), which ishereby incorporated by reference. Generally however, any plasmid orvector may be used as long as it is replicable and viable in a host.

[0095] In another embodiment, the construct is an expression vectorwhich also comprises regulatory sequences operably linked to thesequence of interest, to direct mRNA synthesis and polypeptideproduction. Regulatory sequences known to operate in prokaryotic and/oreukaryotic cells include inducible and non-inducible promoters forregulating mRNA transcription, ribosome binding sited for translationinitiation, stop codons for translation termination and transcriptionterminators and/or polyadenylation signals. In addition, an expressionvector may include appropriate sequences for amplifying expression.

[0096] Promoter regions may be selected from any desired gene.Particular named bacterial promoters include lacZ, gpt, lambda P_(R),lambda P_(L), trc, trp, ermE and its derivatives such as ermEP1ΔTGG,also known in the art as ermE*, (Bibb, M. J., et at., MolecularMicrobiology, 14(3): 533-545 (1994)), melCl, and actII (C. M. Kao, etat., Science, 265: 509-512 (1994)). Eukaryotic promoters includecytomegalovirus (CMV) immediate early, herpes simplex virus (HSV)thymidine kinase, early and late SV40, LTRs from retroviruses, mousemetallothionein-I, prion protein and neuronal specific enolase (NSE).Selection of the appropriate promoter is well within the level ofordinary skill in the art. In addition, a recombinant expression vectorwill include all origin to replication and selectable marker (such as agene conferring resistance to an antibiotic (eg. neomycin,chloramphenicol, ampicillin, or thiostrepton) or a reporter gene (eg.luciferase)) which permit selection of stably transformed or transfectedhost cells.

[0097] In any expression vector, a heterologous structural sequence(i.e. a polynucleotide of the present invention) is assembled inappropriate phase with translation initiation and termination sequences.Optionally, the hetelologous sequence will encode a fusion proteinincluding an N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product.

[0098] Eukaryotic expression vectors will also generally comprise anorigin of replication, a suitable promoter operably linked to a sequenceof interest and also any necessary translation enhancing sequence,polyadenylation site, transcriptional termination sequences, and 5′flanking nontranscribed sequences. DNA sequences derived from the SV40viral genome, for example, SV40 origin, early promoter, enhancer, andpolyadenylation sites may be used to provide the required geneticelements. Such vectors may also include an enhancer sequence to increasetranscription of a gene. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription rate. Examples include the SV40 enhancer on the late sideof the replication origin (bp 100 to 270), a cytomegalovirus earlypromoter enhancer, a polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers.

[0099] i. Vector construction

[0100] The appropriate DNA sequence may be inserted into a vector by avariety of procedures. Generally, site-specific DNA cleavage isperformed by treating the DNA with suitable restriction enzymes underconditions which are generally specified by the manufacturer of thesecommercially available enzymes. Usually, about 1 microgram (μg) ofplasmid or DNA sequence is cleaved 1 unit of enzynme in about 20microliters (μL) of buffer solution by incubation at 37° C. for 1 to 2hours. After incubation with the restriction enzyme, protein can beremoved by phenol/chloroform extraction and the DNA recovered byprecipitation with ethanol. The cleaved fragments may be separated usingpolyacrylamide or agarose gel electrophoresis, according to methodsknown by the routine practitioner. (See Mantiatis et al., Supra).

[0101] Ligations are performed using standard buffer and temperatureconditions and with a ligase (such as T4 DNA ligase) and ATP. Sticky endligations require less ATP and less ligase than blunt end ligations.Vector fragments maybe treated with bacterial alkaline plosphatase (BAP)or calf intestinal alkaline phosphaltase (CIAP) to remove the5′-phosphate and thus prevent religation of the vector. Ligationmixtures are transformed into suitable cloning hosts such as E. coli andsuccessful transformants selected by methods including antibioticresistance, and then screened for the correct construct.

[0102] ii. Transformation/Transfection

[0103] Transformation or transfection of an appropriate host with aconstruct of the invention, such that the host produces recombinantpolypeptides, may also be performed in a variety of ways. For example, aconstruct may be introduced into a host cell by calcium chloride orpolyethylene glycol transformation, lithium chloride or calciumphosphate transfection, DEAE-Dextran mediated transfection, orelectroporation. These and other methods for transforming/tranisfectinghost cells are well known to routine practitioners (see L. Davis et al.,“Basic Methods in Molecular Biology”, 2nd edition, Appleton and Lang,Paramount Publishing, East Norwalk, Conn. (1994) and D. A. Hopwood etal., Genetic Manipulation of Streptomyces: a laboratory manual, The JohnInnes Foundation, Norwich, England (1985)).

[0104] b. Host Cells

[0105] In one embodiment, the present invention provides host cellscontaining recombinant constructs as described below. In one aspect, ahost cell may be an “intermediate” host which is used to producepolynucleotides of the invention on a large-scale basis (for the purposeof cloninig and/or verifying recombinant polynucleotide sequences, forexample) or as a means to maintain such polynucleotide sequences overtime (i.e. as maintenance or storage strains). A “production” host is ahost cell which is used to produce novel polyketides. The host cell(either intermediate or production) can be a higher eukaryotic cell,such as a mammalian cell, or a lower eukaryotic cell, such as a yeastcell, or a prokaryotic cell, such as a bacterial cell. Lower eukaryoticand prokaryotic cells are preferred intermediate and production hosts.

[0106] Representative examples of appropriate hosts include bacterialcells, such is E. coli, Bacillus subtilis, Saccharopolyspora erythraea,Streptomyces caelestis, Streptomyces hygroscopicus, Streptomycesvenezuelae; and various other species within the genera Arthrobacter,Micromonospora, Nocardia, Pseudomonas, Streptomyces, Straphylococcus,and Saccharopolyspora, although other (of eukaryotic origin) may also beemployed. Additional representative examples of host cells arepolyketide-producing microorganisms (as defined above). The selection ofan appropriate host is deemed to be within the scope of those skilled inthe art from the teachings provided herein.

[0107] Host cells are genetically engineered (transduced, transformed,transfected, conjugated, or electroporate) with the vectors of thisinvention which may be a cloning vector or an expression vector. Theengineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or as a source of a biosynthetic substrate. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0108] V. Novel Polyketides and Methods of Making Novel Polyketides

[0109] The invention also provides novel polyketides, intermediatecompounds thereof, and methods for producing novel polyketides. Themethods utilize the polyketide biosynthetic genes from Sac. erythraea(i.e. the eryA genes) as well as those from other knownpolyketide-producing microorganisms and/or putative polyketide-producingmicroorganisms (i.e. those having nucleotide sequences which hybridizeto known PKS sequences but whose polyketide products are unknown).

[0110] The organization of eryA and the DEBS encoded therefrom (see FIG.1 and FIG. 2) have been described in co-pending U.S. application Ser.No. 07/642,734, filed Jan. 17, 1991, which is incorporated herein byreference in its entirety. As FIG. 2 shows, DEBS is organized inmodules, with each module being responsible for one condensation stepthrough the action of the resident KS, AT and ACP domains within thatmodule wherein an extender unit, methylmalonyl CoA, is added first tothe starter unit, propionyl CoA, and then successively to the growingacyl chain. The precise succession of the elongation steps is dictatedby the order of the six modules: module 1 determines the firstcondensation; module 2, the second; module 3, the third, and so on untilthe sixth condensation step has occurred. In addition, the choice ofextender unit that is incorporated into a growing polyketide chain ateach condensation is determined, in whole or in part, by the AT domainwithin each module. In the case of DEBS, the extender unit incorporatedis always methylmalonate. Thus, as 6-deoxyerythronolide B grows throughsuccessive condensations, two carbons are added to the nascent chain andevery other carbon, starting with the carbon corresponding to C-12 inthe ring, carries a methyl group as a side chain.

[0111] As also seen in FIG. 2, the processing of the growing carbonchain after each condensation is determined by the information withineach module. Thus, β-ketoreduction of the β-keto group generated by thecondensation event takes place after each condensation step except thethird, as determined by the presence of all active KR domain in eachmodule except module 3, whereas dehydration and enoylreduction takeplace after the fourth condensation step, as determined by the presenceof the DH and ER domains in module 4. Once the polyketide chain is fullysynthesized, it is released from the PKS through the action of the TEdomain present at the end of module 6 and cyclizes to form themacrocyclic lactone 6-deoxyerythronolide B which is subsequently actedupon by a series of other enzymes, whose genes reside in theerythromycin cluster of the Sac. erythtraea chromosome (see FIG. 1). Asshown in FIG. 1, erythromycin carries methyl side chains at position 2,4, 6, 8, 10 and 12, through the incorporation of methylmalonate as theextender unit at each step of synthesis of the polyketide moiety.

[0112] In the present invention, novel polyketide molecules of a desiredstructure are produced by introducing specific genetic alterations intoa PKS-encoding sequence in the genome of a polyketide-producinigmicroorganism. Alteration of one or more genes or fragments thereof maybe generated through manipulation of genes residing exclusively within aspecies (i.e. intraspecies alterations), and include not onlymanipulations of genes within a single PKS cluster but also betweendifferent PKS clusters residing within a single strain (as is seen in S.hygroscopicus). Several examples of intraspecies alterations showing themanipulation of genes exclusively within a single PKS (namely, eryA) aredescribed in U.S. application Ser. No. 07/624,734 cited supra.Alternatively, a gene or fragment thereof may be exchanged with aheterologous gene or gene fragment encoding one or more related domainsfrom the PKS of a different polyketide-producing microorganism(interspecies alterations). Several examples of novel polyketidesproduced from exchange of heterologous genes are provided herein.

[0113] Whether the genetic manipulations are performed intraspecies orinterspecies, three types of alterations to a PKS sequence may becarried out: (i) those which affect a module but do not cause the arrestof chain growth (Type I alterations); (ii) those which affect a singlefunction in a module thereby causing the arrest of chain growth (Type IIalterations); and (iii) those which affect an entire module (Type IIIalterations). In one embodiment, Type I alterations are produced byinactivation of domains that specify the functional groups and/or degreeof oxidation found at specific ring positions in the native polyketide.Such domains typically include β-ketoreductases, dehydratases andenoylreductases. For example, an illele corresponding to β-ketoreductaseof module 5 may be mutated by deleting a substantial portion of the DNAencoding the β-ketoredtuctase (thereby producing an inactive domain) andused to replace the wild-type allele in the native strain. Such atransfer results in the production of the novel polyketide5-oxo-5,6-dideoxy-3-∝-mycarosyl erythronolide B.

[0114] In an alternative embodiment, Type I alterations are generated byreplacing at least one domain in a particular PKS with at least onerelated domain from the same or a second PKS. Such related domains mayexist between different polyketide-producing microorganisms (such as forexample, the AT domains of Sac. erythraea, S. venezuelae, S.hygroscopicus, and S. caelestis) or within (a single species (as forexample, the LigAT2 and rapAT1 domains in S. hygroscopicus).

[0115] Ways to identify polyketide synthases, their domains and thefunctional similarity of domains are well-known to those of ordinaryskill in the art. For example, the PKS region of the chromosome of apolyketide or putative polyketide-producing microorganism may beidentified by hybridizing with nucleic acid probes under conditions oflow or high stringency. Hybridization under high stringency conditionsis generally performed in a buffer consisting of 15 mM sodium chlorideand 1.5 mM trisodium citrate (0.1×SSC) with an incubation temperature ofabout 65° C. (see for example, Maniatis, et al. supra). To detect moredistantly related PKS genes, hybridization is performed under lowstringency conditions which include lower temperature incubations and/orthe presence of increased amounts of sodium chloride and trisodiumcitrate (Maniatis, et al. supra). Once identified, the chromosomalregion may be isolated, cloned into a suitable vector and sequenced,using conventional methods or commercial sequencing kits such asSequenase (US Biochemical Corp, Cleveland, Ohio). Methods for isolatingand cloning chromosomal DNA are also well known in the art (Maniatis, etal. supra). An aminio icid sequence may then be deduced from the DNAsequence and a comparison made of the unknown amino acid sequence tothat of one or more polypeptides involved in polyketide biosynthesis.Two amino acid sequences showing at least about 20% and more preferablyabout 25% identity and having conserved active site residues or motifsare considered to specify functionally similar or equivalent PKSdomains. Having identified such domains, the number and composition ofmodules as well as the arrangement of modules within particular ORFs canbe determined.

[0116] In the case where the newly defined PKS produces a polyketide ofknown structure, the β-carbonyl processing and types of side chainmoieties and their positioning on the polyketide backbone can becorrelated to specific domains within modules. Because modules areestablished linearly within ORFs, this correlation also allows one todetermine the order of modular activity (i.e. which module catalyzeswhich condensation step) in the PKS. For example, the β-carbonylprocessing and types of side chain moieties in the polyketide generatesa pattern of chemical groups that can be correlated to a pattern ofdomains within an ORF. Based on the specific type of side chain moietyat a given carbon, one can then predict the particular substrateutilized by that module's AT domain.

[0117] In the case where the polyketide structure is unknown,theoretically, comparative sequence analysis alone may be used topredict the substrate specificity of an AT domain. To accomplish this,at least two and preferably, three or more sequences known or predictedto specify a particular substrate can be compared to determine one ormore conserved or consensus motifs unique to that family of Ats. Anunknown At having such motifs can then be assigned to a particularfamily.

[0118] Alternatively, comparative anatyses caln he performed usingcomputer programs which group AT domains based on primary amino acidsequence similarity or phylogenetic relationships. For example,comparative analyses were made of the amino acid sequences of the ATdomains in DEBS with corresponding AT domains in the PKS for rapamycinto determine whether the extender unit used by a particular AT domain,(either malonate or methylmalonate), correlated with the degree ofsequence identity between these domains. Rapamycin is a large polyketidethat is assembled through 14 condensation events; the rapamycin PKSpossesses 14 AT domains whose sequences were deduced from knownnucleotide sequences (Aparicio et at. Gene 169:9-16 (1996)). Amino acidsequence comparisons of the 14 AT domains of the rapamycin PKS with eachother and with the 6 AT domains from DEBS, showed that the AT domainsfell into two distinct groupings in which the rapamycin AT domains frommodules 1, 3, 4, 6, 7, 10 and 13 clustered with the 6 erythromycin ATdomains and the rapamycin AT domains in modules 2, 5, 8, 9, 11, 12 and14 formed a separate cluster (Haydock et al. FEBS Letts. 374:246-248(1995)). Examination of the polyketide structure of rapamycin indicatedthat methyl side chains were at positions on the lactone ringcorresponding to condensation steps 1, 3, 4, 6, 7, 10 and 13, whichsuggested that methylmalonate was used as the extender unit duringsynthesis of these sections of the acyl chain; protons at the positionsof the lactone ring corresponing to condensations steps 2, 5, 8, 9, 11,12 and 14 suggested that malonate was utilized as the extender unitduring synthesis of these sections. Two additional AT domains describedherein, ligAT2 and venAT, were also found to cluster with the putativemalonate AT domains from the rapamycin PKS (FIG. 3). Having predictedthat AT domains from rap modules 2, 5, 8, 9, 11, 12 or 14, as well asligAT2 and venAT, specify malonate as extender units, the DNA encodingsuch domains could be isolated, cloned and used to replace the DNAencoding one or more AT domains in a PKS such as DEBS, in order togenerate novel polyketides.

[0119] The techniques for determining the amino acid sequence“similarity” are well-known in the art. In general, when two or morepolypeptides are aligned with one another, their sequence similarityrefers to the amino acids at corresponding positions within eachpolypeptide sequence that are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. In general, the term “identity” refers to anexact nucleotide to nucleotide or amino acid to amino acidcorrespondence at a given position of two polynucleotides or polypeptidesequences, respectively. Two amino acid sequences (or for that matter,two or more polynucleotide sequences) can be compared by determiningtheir “percent identity.” The programs availabile in the WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup (GCG), Madison, Wis.), for example, the GAP program, are capableof calculating both the identity between two polynucleotides and theidentity and similarity between two polypeptide sequences, respectively.Other programs for calculating and displaying similarity betweensequences are known in the art. For example, the Growtree program (GCG,Madison, Wis.) creates a phylogenetic tree wherein the most closelyrelated sequences are clustered and joined by the shortest lines. Thistree is derived from a matrix created by the program Distances (GCG,Madison, Wis.) which calculates pairwise relationships within a group ofaligned sequences.

[0120] In a preferred embodiment, novel polyketide molecules of desiredstructure are produced by the replacement of at least one ATdomain-encoding fragment of DNA of the Sac. erythraea chromosome with atleast one heterologous AT domain-encoding fragment of DNA from anotherPKS cluster to yield novel polyketide compounds which are derivatives of6-deoxyerythronolide B, erythronolide B, 3-α-L-mycarosylerythronolide B,or erythromycins A, B, C and D. Such derivatives are compounds whereinmethyl (—Me) side chains at one or more positions of the macrocyliclactone ring are replaced by substituents independently selected fromthe group consisting of (a) —H; (b) ethyl group (-Et); (c) hydroxylgroup (—OH) and (d) allyl group (—Al). In a particularly preferredembodiment, a method is provided for the genetic modification oferythromycin-producing microorganisms which enables them to produce thenovel compounds 12-desmethyl-12-deoxyerythromycin A,10-desmethylerythromycin A, 10-desmethyl-12-deoxyerythromycin A, or6-desmethyl-6-ethylerythromycin A. The compounds12-desmethyl-12-deoxyerythromycin A, 10-desmethylerythromycin A,10-desmethyl-12-deoxyerythromycin A, and 6-desmethyl-6-ethylerythromycinA are represented by the structural formulae:

[0121] The general scheme for producing such polyketides is outlined inFIG. 4a and FIG. 4b). In the preferred embodiment, heterologous DNAfragments encoding related AT domains are introduced into the Sac.erythraea chromosome by a two-step method termed gene replacement.

[0122] In the first step of gene replacement, an integration vector isconstructed through a multi-step cloning approach that places aheterologous gene or fragment thereof between two segments of DNA havingsequences which are identical to those that immediately border (on eachside) the resident polynucleotide sequence to be replaced. Constructionof such a vector may be achieved by any means known to those of ordinaryskill in art. For example, nucleotide sequences which flank the gene tobe replaced can be generated by PCR amplification using chromosonal DNAas template and primers which hybridize to the chromosomal sequencesimmediately upstream and downstream of the flanking sequences ofinterest. The length of the flanking sequences is not critical to thepractice of the invention but preferably is about 20-5000 base pairs(bp), more preferably about 100-5000 bp, and even more preferably about500-5000 bp. A most preferred length of flanking sequence is about750-1500 bp. Primers used for such amplifications may also compriseconvenient restriction sites to facilitate cloning of the amplifiedsequences into suitable preparative vectors, to facilitate insertion ofthe heterologous sequence of interest between the flanking sequencesand/or to facilitate subcloning of the entire group of sequences(5′-flanking region/heterologous polynucleotide sequence ofinterest/flanking region-3′) into suitable vectors for integration. Thedesired heterologous polynucleotide sequences mnay be generated in alike manner.

[0123] The integration vectors are constructed to also comprise afragment of DNA containing at least one origin of replication that isfunctional in an intermediate host but is non-functional or poorlyfunctional in the production host. The vectors further comprise one ormore fragments of DNA conferring resistance to an antibiotic, of whichat least one functions in the intermediate host and at least onefunctions in the production host. Preferred integration vectors comprisethe ColE1 and pIJ101 origins of replication, as found in plasmid pCS5(J. Vara et al., J. Bacteriol. 171:5972-5991 (1989)). A particularlypreferred vector carries a DNA fragment conferring resistance tothiostrepton and ampicillin. However, those skilled in the artunderstand that the particular antibiotic resistance genes and originsof replication identified above are necessary only inasmuch as theyallow for the generation and selection of the desired recombinantplasmids and host cells. Other markers and origins of replication mayalso be used in the practice of the invention.

[0124] When the resident domains of a PKS are functional components oflarge multifunctional polypeptides, care must be taken in theconstruction of the integration plasmid so that the heterologous DNAfragment encoding the heterologous AT domain is positioned in thecorrect orientation and reading frame to its flanking DNA segments sothat upon translation from the beginning of the coding sequence, anenzymatically functional protein is produced. The correct positioningbecomes immediately apparent from knowledge of the nucleotide sequencesof the host PKS genes and the heterologous genes used for genereplacement.

[0125] In the second step, each of the integration vectors carrying arelated gene or fragment thereof is independently introduced into a hoststrain and recombination between each of the genomic fragments in theintegration plasmid and its corresponding homologous fragment in thehost strain chromosome is allowed to occur. This procedure results inthe exchange of the resident AT-encoding DNA in the chromosome for itsheterologous counterpart. The general scheme for gene replacement byhomologous recombination is outlined in FIG. 5. Procedures to introduceDNA into polyketide-producing microorganisms and to facilitatehomologous recombination are described herein. However, those skilled inthe art understand that alternative procedures for introducing DNA intoa polyketide-producing microorganism, such as electroporation,transduction, or conjugation, are well known and may also be used in thepractice of the invention. Procedures for cultivatingpolyketide-producing microorganisms, as well as methods to recover novelpolyketides produced from modified strains, to purify such compounds andto confirm the identity of those compounds (such as by mass spectrometryor NMR) are well-known to those of ordinary skill in the art.

[0126] Although the present invention is described in the Examples thatfollow in terms of preferred embodiments, they are not to be regarded aslimiting the scope of the invention. The descriptions that follow serveto illustrate the principles and methodologies involved in creatingnovel derivatives of erythromycin. Whereas the examples below describethe replacement of the Sac. erythraea AT1, AT2, and AT4-encoding DNAfragments with a heterologous DNA fragment which encodes either an ATdomain that specifies incorporation of malonate (malonate-AT) or an ATdomain that specifies incorporation of ethylmalonate (ethylmalonate-AT),those skilled in the art understand that one or more fragments ofheterologous DNA encoding malonate, ethylmalonate, allylmalonate, and/orhydroxymalonate (tartronate)-AT domains can be used to replace the otherAT-encoding DNA fragments of the erythromycin PKS in Sac. erythraea toresult in the production of other novel erythromycin derivatives. Forexample, novel erythromycins produced when resident AT-encoding DNAfragments in the erythromycin PKS (eryPKS) are independently replacedwith heterologous DNA fragments specifying malonate and/or ethylmalonateas the extender unit are shown in Table 1.

[0127] In particular, those skilled in the art understand that followingthe methods described herein for replacement of a single residentAT-encoding DNA fragment in the erylPKS, replacements of two residentAT-encoding DNA fragments with heterologous DNA fragments (specifyingmalonate, ethylmalonate, allylmalonate, and/or hydroxymalonate-ATdomains) in stepwise fashion are also possible and result in theformation of novel disubstituted erythromycins. Similarly,trisubstituted erythromycins, tetrasubstitued erythromycins,pentasubstitued erythromycins and hexasubstitued erythromycins can alsobe made by replacement of three, four, five and six resident AT-encodingDNA fragments in the erylPKS, respectively, with heterologousAT-encoding DNA fragments as described herein. Therefore, allsubstitutions of AT-encoding DNA fragments in the eryPKS withheterologous AT-encoding DNA fragments (yielding all varieties ofproton, ethyl, allyl, and hydroxyl substituted erythromycin derivatives)are within the scope of the present invention. Examples of compoundsproduced by such replacements include but are not limited to those shownin Table 1 below. TABLE 1 Structures from Changes at Side ChainPositions

R₁ R₂ R₃ R₄ R₅ R₆ Name A. Single Changes H Me Me Me Me Me12-Desmethylerythromycin A Et Me Me Me Me Me12-Desinethyl-12-ethylerythromycin A Me H Me Me Me Me10-Desmethylerythromycin A Me Et Me Me Me Me10-Desmehyl-10-ethylerythromycin A Me Me H Me Me Me8-Desmethylerythromycin A Me Me Et Me Me Me8-Desmethyl-8-ethylerythromycin A Me Me Me H Me Me6-Desmethylerythromycin A Me Me Me Et Me Me6-Desmethyl-6-ethylerythromycin A Me Me Me Me H Me4-Desmethylerythromycin A Me Me Me Me Et Me4-Desmethyl-4-ethylerythromycin A Me Me Me Me Me H2-Desmethylerythromycin A Me Me Me Me Me Et2-Desmethyl-2-ethylerythromycin A B. Two Changes H Me Me Me Me Et2,12-Didesmethyl-2-ethylerythromycin A H Me Me Me Et Me4,12-Didesmethyl-4-ethylerythromycin A H Me Me Et Me Me6,12-Didesmethyl-6-ethylerythromycin A H Me Et Me Me Me8,12-Didesmethyl-8-ethylerythromycin A H Et Me Me ME Me10,12-Didesmethyl-10-ethylerythromycin A H Me Me Me Me H2,12-Didesmethylerythromycin A H Me Me Me H Me4,12-Didesmethylerythromycin A H Me Me H Me Me6,12-Didesmethylerythromycin A H Me H Me Me Me8,12-Didesmethylerythromycin A H H Me Me Me Me10,12-Didesmethylerythromycin A Me H Me Me Me Et2,10-Didesmethyl-2-ethylerythromycin A Me H Me Me Et Me4,10-Didesmethyl-4-ethylerythromycin A Me H Me Et Me Me6,10-Didesmethyl-6-ethylerythromycin A Me H Et Me Me Me8,10-Didesmethyl-8-ethylerythromycin A Me H Me Me Me H2,10-Didesmethylerythromycin A Me H Me Me H Me4,10-Didesmethylerythromycin A Me H Me H Me Me6,10-Didesmethylerythromycin A Me H H Me Me Me8,10-Didesmethylerythromycin A Me Me H Me Me Et2,8-Didesmethyl-2-ethylerythromycin A Me Me H Me Et Me4,8-Didesmethyl-4-ethylerythromycin A Me Me H Et Me Me6,8-Didesmethyl-6-ethylerythromycin A Me Me H Me Me H2,8-Didesmethylerythromycin A Me Me H Me H Me4,8-Didesmethylerythromycin A Me Me H H Me Me6,8-Didesmethylerythromycin A Me Me Me H Me Et2,6-Didesmethyl-2-ethylerythromycin A Me Me Me H Et Me4,6-Didesmethyl-4-ethylerythromycin A Me Me Me H Me H2,6-Didesmethylerythromycin A Me Me Me H H Me4,6-Didesmethylerythromycin A Me Me Me Me H Et2,4,-Didesmethyl-2-ethylerythromycin A Me Me Me Me H H2,4,-Didesmethylerythromycin A Et Me Me Me Me Et2,12-Didesmethyl-2,12-diethylerythromycin A Et Me Me Me Et Me4,12-Didesmethyl-4,12-diethylerythromycin A Et Me Me Et Me Me6,12-Didesmethyl-6,12-diethylerythromycin A Et Me Et Me Me Me8,12-Didesmethyl-8,12-diethylerythromycin A Et Et Me Me Me Me10,12-Didesmethyl-10,12-diethylerythromycin A Et Me Me Me Me H2,12-Didesmethyl-12-ethylerythromycin A Et Me Me Me H Me4,12-Didesmethyl-12-ethylerythromycin A Et Me Me H Me Me6,12-Didesmethyl-12-ethylerythromycin A Et Me H Me Me Me8,12-Didesmethyl-12-ethylerythromycin A Et H Me Me Me Me10,12-Didesmethyl-12-ethylerythromycin A Me Et Me Me Me Et2,10-Didesmethyl-2,10-diethylerythromycin A Me Et Me Me Et Me4,10-Didesmethyl-4,10-diethylerythromycin A Me Et Me Et Me Me6,10-Didesmethyl-6,10-diethylerythromycin A Me Et Et Me Me Me8,10-Didesmethyl-8,10-diethylerythromycin A Me Et Me Me Me H2,10-Didesmethyl-10-ethylerythromycin A Me Et Me Me H Me4,10-Didesmethyl-10-ethylerythromycin A Me Et Me H Me Me6,10-Didesmethyl-10-ethylerythromycin A Me Et H Me Me Me8,10-Didesmethyl-10-ethylerythromycin A Me Me Et Me Me Et2,8-Didesmethyl-2,8-diethylerythromycin A Me Me Et Me Et Me4,8-Didesmethyl-4,8-diethylerythromycin A Me Me Et Et Me Me6,8-Didesmethyl-6,8-diethylerythromycin A Me Me Et Me Me H2,8-Didesmethyl-8-ethylerythromycin A Me Me Et Me H Me4,8-Didesmethyl-8-ethylerythromycin Me Me Et H Me Me6,8-Didesmethyl-8-ethylerythromycin Me Me Me Et Me Et2,6-Didesmethyl-2,6-diethylerythromycin A Me Me Me Et Et Me4,6-Didesmethyl-4,6-diethylerythromycin A Me Me Me Et Me H2,6-Didesmethyl-6-ethylerythromycin A Me Me Me Et H Me4,6-Didesmethyl-6-ethylerythromycin Me Me Me Me Et Et2,4-Didesmethyl-2,4-diethylerythromycin A Me Me Me Me Et H2,4-Didesmethyl-4-ethylerythromycin A C. Three Changes H H Me Me Me Et2,10,12-Tridesmethyl-2-Ethylerythromycin A H H Me Me Me H2,10,12-Tridesmethylerythromycin A H H Me Me Et Me4,10,12-Tridesmethyl-4-Ethylerythromycin A H H Me Me H Me4,10,12-Tridesmethylerythromycin A H H Me Et Me Me6,10,12-Tridesmethyl-6-Ethylerythromycin A H H Me H Me Me6,10,12-Tridesmethylerythromycin A H H Et Me Me Me8,10,12-Tridesmethyl-8-ethylerythromycin A H H H Me Me Me8,10,12-Tridesmethylerythromycin A Et H Me Me Me Et2,10,12-Tridesmethyl-2,12,-diethylerythromycin A Et H Me Me Me H2,10,12-Tridesmethyl-12-ethylerythromycin A Et H Me Me Et Me4,10,12-Tridesmethyl-4,12-diethylerythromycin A Et H Me Me H Me4,10,12-Tridesmethyl-12-ethylerythromycin A Et H Me Et Me Me6,10,12-Tridesmethyl-6,12-diethylerythromycin A Et H Me H Me Me6,10,12-Tridesmethyl-12-ethylerythromycin A Et H Et Me Me Me8,10,12-Tridesmethyl-8,12-diethylerythromycin A Et H H Me Me Me8,10,12_Tridesmethyl-12-ethylerythromycin A H Et Me Me Me Et2,10,12-Tridesmethyl-2,10-diethylerythromycin A H Et Me Me Me H2,10,12-Tridesmethyl-10-ethylerythromycin A H Et Me Me Et Me4,10,12-Tridesmethyl-4,10-diethylerythromycin A H Et Me Me H Me4,10,12-Tridesmethyl-10-ethylerythromycin A H Et Me Et Me Me6,10,12-Tridesmethyl-6,10-diethylerythromycin A H Et Me H Me Me6,10,12-Tridesmethyl-10-ethylerythromycin A H Et Et Me Me Me8,10,12-Tridesmethyl-8,10-diethylerythromycin A H Et H Me Me Me8,10,12-Tridesmethyl-10-ethylerythromycin A Et Et Me Me Me Et2,10,12-Tridesmethyl-2,10,12-triethylerythromycin A Et Et Me Me Me H2,10,12-Tridesmethyl-10,12-diethylerythromycin A Et Et Me Me Et Me4,10,12-Tridesmethyl-4,10,12-triethylerythromycin A Et Et Me Me H Me4,10,12-Tridesmethyl-10,12,-diethylerythromycin A Et Et Me Et Me Me6,10,12-Tridesmethyl-6,10,12-triethylerythromycin A Et Et Me H Me Me6,10,12-Tridesmethyl-10,12-diethylerythromycin A Et Et Et Me Me Me8,10,12-Tridesmethyl-8,10,12-triethylerythromycin A Et Et H Me Me Me8,10,12-Tridesmethyl-10,12-diethylerythromycin A H Me H Me Me Et2,8,12-Tridesmethyl-2-ethylerythromycin A H Me H Me Me H2,8,12-Tridesmethylerythromycin A H Me H Me Et Me4,8,12-Tridesmethyl-4-ethylerythromycin A H Me H Me H Me4,8,12-Tridesmethylerythromycin A H Me H Et Me Me6,8,12-Tridesmethyl-6-ethylerythromycin A H Me H H Me Me6,8,12-Tridesmethylerythromycin A Et Me H Me Me Et2,8,12-Tridesmethyl-2,12-diethylerythromycin A Et Me H Me Me H2,8,12-Tridesmethyl-12-ethylerythromycin A Et Me H Me Et Me4,8,12-Tridesmethyl-4,12-diethylerythromycin A Et Me H Me H Me4,8,12-Tridesmethyl-12-ethylerythromycin A Et Me H Et Me Me6,8,12-Tridesmethyl-6,12-diethylerythromycin A Et Me H H Me Me6,8,12-Tridesmethyl-12-ethylerythromycin A H Me Et Me Me Et2,8,12-Tridesmethyl-2,8-diethylerythromycin A H Me Et Me Me H2,8,12-Tridesmethyl-8-ethylerythromycin A H Me Et Me Et Me4,8,12-Tridesmethyl-4,8-diethylerythromycin A H Me Et Me H Me4,8,12-Tridesmethyl-8-ethylerythromycin A H Me Et Et Me Me6,8,12-Tridesmethyl-6,8-diethylerythromycin A H Me Et H Me Me6,8,12-Tridesmethyl-8-ethylerythromycin A Et Me Et Me Me Et2,8,12-Tridesmethyl-2,8,12-triethylerythromycin A Et Me Et Me Me H2,8,12-Tridesmethyl-8,12-diethylerythromycin A Et Me Et Me Et Me4,8,12-Tridesmethyl-4,8,12-triethylerythromycin A Et Me Et Me H Me4,8,12-Tridesmethyl-8,12-diethylerythromycin A Et Me Et Et Me Me6,8,12-Tridesmethyl-6,8,12-triethylerythromycin A Et Me Et H Me Me6,8,12-Tridesmethyl-8,12-diethylerythromycin A H Me Me H Me Et2,6,12-Tridesmethyl-2-ethylerythromycin A H Me Me H Me H2,6,12-Tidesmethylerythromycin A H Me Me H Et Me4,6,12-Tridesmethyl-4-ethylerythromycin A H Me Me H H Me4,6,12-Tridesmethylerythromycin A Et Me Me H Me Et2,6,12-Tridesmethyl-2,12-diethylerythromycin A Et Me Me H Me H2,6,12-Tridesmethyl-12-ethylerythromycin A Et Me Me H Et Me4,6,12-Tridesmethyl-4,12-diethylerythromycin A Et Me Me H H Me4,6,12-Tridesmethyl-12-ethylerythromycin A H Me Me Et Me Et2,6,12-Tridesmethyl-2,6-diethylerythromycin A H Me Me Et Me H2,6,12-Tridesmethyl-6,-ethylerythromycin A H Me Me Et Et Me4,6,12-Tridesmethyl-4,6-diethylerythromycin A H Me Me Et H Me4,6,12-Tridesmethyl-6-ethylerythromycin A Et Me Me Et Me Et2,6,12-Tridesmethyl-2,6,12-triethylerythromycin A Et Me Me Et Me H2,6,12-Tridesmethyl-6,12-diethylerythromycin A Et Me Me Et Et Me4,6,12-Tridesmethyl-4,6,12-triethylerythromycin A Et Me Me Et H Me4,6,12-Tridesmethyl-6,12-diethylerythromycin A H Me Me Me H Et2,4,12-Tridesmethyl-2-ethylerythromycin A H Me Me Me H H2,4,12-Tridesmethylerythromycin A Et Me Me Me H Et2,4,12-Tridesmethyl-2,12-diethylerythromycin A Et Me Me Me H H2,4,12-Tridesmethyl-12-ethylerythromycin A H Me Me Me Et Et2,4,12-Tridesmethyl-2,4-diethylerythromycin A H Me Me Me Et H2,4,12-Tridesmethyl-4-ethylerythromycin A Et Me Me Me Et Et2,4,12-Tridesmethyl-2,4,12-triethylerythromycin A Et Me Me Me Et H2,4,12-Tridesmethyl-2,12-diethylerythromycin A Me H H Me Me Et2,8,10-Tridesmethyl-2-ethylerythromycin A Me H H Me Me H2,8,10-Tridesmethylerythromycin A Me H H Me Et Me4,8,10-Tridesmethyl-4-ethylerythromycin A Me H H Me H Me4,8,10-Tridesmethylerythromycin A Me H H Et Me Me6,8,10-Tridesmethyl-6-ethylerythromycin A Me H H H Me Me6,8,10-Tridesmethylerythromycin A Me Et H Me Me Et2,8,10-Tridesmethyl-2,10-diethylerythromycin A Me Et H Me Me H2,8,10-Tridesmethyl-10-ethylerythromycin A Me Et H Me Et Me4,8,10-Tridesmethyl-4,10-diethylerythromycin A Me Et H Me H Me4,8,10-Tridesmethyl-10-ethylerythromycin A Me Et H Et Me Me6,8,10-Tridesmethyl-6,10-diethylerythromycin A Me Et H H Me Me6,8,10-Tridesmethyl-10-ethylerythromycin A Me H Et Me Me Et2,8,10-Tridesmethyl-2,8-diethylerythromycin A Me H Et Me Me H2,8,10-Tridesmethyl-8-ethylerythromycin A Me H Et Me Et Me4,8,10-Tridesmethyl-4,8-diethylerythromycin A Me H Et Me H Me4,8,10-Tridesmethyl-8-ethylerythromycin A Me H Et Et Me Me6,8,10-Tridesmethyl-6,8-diethylerythromycin A Me H Et H Me Me6,8,10-Tridesmethyl-8-ethylerythromycin A Me Et Et Me Me Et2,8,10-Tridesmethyl-2,8,10-triethylerythromycin A Me Et Et Me Me H2,8,10-Tridesmethyl-8,10-diethylerythromycin A Me Et Et Me Et Me4,8,10-Tridesmethyl-4,8,10-triethylerythromycin A Me Et Et Me H Me4,8,10-Tridesmethyl-8-10-diethylerythromycin A Me Et Et Et Me Me6,8,10-Tridesmethyl-6,8,10-triethylerythromycin A Me Et Et H Me Me6,8,10-Tridesmethyl-8,10-diethylerythromycin A Me H Me H Me Et2,6,10-Tridesmethyl-2-ethylerythromycin A Me H Me H Me H2,6,10-Tridesmethylerythromycin A Me H Me H Et Me4,6,10-Tridesmethyl-4-ethylerythromycin A Me H Me H H Me4,6,10-Tridesmethylerythromycin A Me Et Me H Me Et2,6,10-Tridesmethyl-2,1--diethylerythromycin A Me Et Me H Me H2,6,10-Tridesmethyl-10-ethylerythromycin A Me Et Me H Et Me4,6,10-Tridesmethyl-4,10-diethylerythromycin A Me Et Me H H Me4,6,10-Tridesmethyl-10-ethylerythromycin A Me H Me Et Me Et2,6,10-Tridesmethyl-2,6-diethylerythromycin A Me H Me Et Me H2,6,10-Tridesmethyl-6-ethylerythromycin A Me H Me Et Et Me4,6,10-Tridesmethyl-4,6,diethylerythromycin A Me H Me Et H Me4,6,10-Tridesmethyl-6-ethylerythromycin A Me Et Me Et Me Et2,6,10-Tridesmethyl-2,6,10-triethylerythromycin A Me Et Me Et Me H2,6,10-Tridesmethyl-6,10-diethylerythromycin A Me Et Me Et Et Me4,6,10-Tridesmethyl-4,6,10-triethylerythromycin A Me Et Me Et H Me4,6,10-Tridesmethyl-6,10-diethylerythromycin A Me H Me Me H Et2,4,10-Tridesmethyl-2-ethylerythromycin A Me H Me Me H H2,4,10-Tridesmethylerythromycin A Me Et Me Me H Et2,4,10-Tridesmethyl-2,10-diethylerythromycin A Me Et Me Me H H2,4,10-Tridesmethyl-10-ethylerythromycin A Me H Me Me Et Et2,4,10-Tridesmethyl-2,4-diethylerythromycin A Me H Me Me Et H2,4,10-Tridesmethyl-4-ethylerythromycin A Me Et Me Me Et Et2,4,10-Tridesmethyl-2,4,10-triethylerythromycin A Me Et Me Me Et H2,4,10-Tridesmethyl-4,10-diethylerythromycin A Me Me H H Me Et2,6,8-Tridesmethyl-2-ethylerythromycin A Me Me H H Me H2,6,8-Tridesmethylerythromycin A Me Me H H Et Me4,6,8-Tridesmethyl-4-ethylerythromycin A Me Me H H H Me4,6,8-Tridesmethylerythromycin A Me Me Et H Me Et2,6,8-Tridesmethyl-2,8-diethylerythromycin A Me Me Et H Me H2,6,8-Tridesmethyl-8-ethylerythromycin A Me Me Et H Et Me4,6,8-Tridesmethyl-4,8-diethylerythromycin A Me Me Et H H Me4,6,8-Tridesmethyl-8-ethylerythromycin A Me Me H Et Me Et2,6,8-Tridesmethyl-2,6-diethylerythromycin A Me Me H Et Me H2,6,8-Tridesmethyl-6-ethylerythromycin A Me Me H Et Et Me4,6,8-Tridesmethyl-4,6-diethylerythromycin A Me Me H Et H Me4,6,8-Tridesmethyl-6-ethylerythromycin A Me Me Et Et Me Et2,6,8-Tridesmethyl-2,6,8-triethylerythromycin A Me Me Et Et Me H2,6,8-Tridesmethyl-6,8-diethylerythromycin A Me Me Et Et Et Me4,6,8-Tridesmethyl-4,6,8-triethylerythromycin A Me Me Et Et H Me4,6,8-Tridesmethyl-6,8-triethylerythromycin A Me Me H Me H Et2,4,8-Tridesmethyl-2-ethylerythromycin A Me Me H Me H H2,4,8-Tridesmethylerythromycin A Me Me Et Me H Et2,4,8-Tridesmethyl-2,8-diethylerythromycin A Me Me Et Me H H2,4,8-Tridesmethyl-8-ethylerythromycin A Me Me H Me Et Et2,4,8-Tridesmethyl-2,4-diiethylerythromycin A Me Me H Me Et H2,4,8-Tridesmethyl-4-ethylerythromycin A Me Me Et Me Et Et2,4,8-Tridesmethyl-2,4,8-triethylerythromycin A Me Me Et Me Et H2,4,8-Tridesmethyl-4,8-diethylerythromycin A Me Me Me H H Et2,4,6-Tridesmethyl-2-ethylerythromycin A Me Me Me H H H2,4,6-Tridesmethylerythromycin A Me Me Me Et H Et2,4,6-Tridesmethyl-2,6-diethylerythromycin A Me Me Me Et H H2,4,6-Tridesmethyl-6-ethyl erythromycin A Me Me Me H Et Et2,4,6-Tridesmethyl-2,4-diethyl erythromycin A Me Me Me H Et H2,4,6-Tridesmethyl-4-ethyl erythromycin A Me Me Me Et Et Et2,4,6-Tridesmethyl-2,4,6-triethyl erythromycin A Me Me Me Et Et H2,4,6-Tridesmethyl-4,6-diethyl erythromycin A D. Four Changes H H H MeMe Et 2,8,10,12-Tetradesmethyl-2-ethylerythromycin A H H H Me Me H2,8,10,12-Tetradesmethylerythromycin A H H H Me Et Me4,8,10,12-Tetradesmethyl-4-ethylerythromycin A H H H Me H Me4,8,10,12-Tetradesmethylerythromycin A H H H Et Me Me6,8,10,12-Tetradesmethyl-6-ethylerythromycin A H H H H Me Me6,8,10,12-Tetradesmethylerythromycin A H Et H Me Me Et2,6,10,12-Tetradesmethyl-2,10-diethylerythromycin A H Et H Me Me H2,6,10,12-Tetradesmethyl-10-ethylerythromycin A H Et H Me Et Me4,8,10,12-Tetradesmethyl-4,10-diethylerythromycin A H Et H Me H Me4,8,10,12-Tetradesmethyl-10-ethylerythromycin A H Et H Et Me Me6,8,10,12-Tetradesmethyl-6,10-diethylerythromycin A H Et H H Me Me6,8,10,12-Tetradesmethyl-10-ethylerythromycin A H H Et Me Me Et2,8,10,12-Tetradesmethyl-2,8-diethylerythromycin A H H Et Me Me H2,8,10,12-Tetradesmethyl-8-ethylerythromycin A H H Et Me Et Me4,8,10,12-Tetradesmethyl-4,8-diethylerythromycin A H H Et Me H Me4,8,10,12-Tetradesmethyl-8-ethylerythromycin A H H Et Et Me Me6,8,10,12-Tetradesmethyl-6,8-diethylerythromycin A H H Et H Me Me6,8,10,12-Tetradesmethyl-8-ethylerythromycin A H Et Et Me Me Et2,6,10,12-Tetradesmethyl-2,8,10-triethylerythromycin A H Et Et Me Me H2,6,10,12-Tetradesmethyl-8,10-diethylerythromycin A H Et Et Me Et Me4,8,10,12-Tetradesmethyl-4,8,10-triethylerythromycin A H Et Et Me H Me4,8,10,12-Tetradesmethyl-8,10-diethylerythromycin A H Et Et Et Me Me6,8,10,12-Tetradesmethyl-6,8,10-triethylerythromycin A H Et Et H Me Me6,8,10,12-Tetradesmethyl-8,10-diethylerythromycin A Et H H Me Me Et2,8,10,12-Tetradesmethyl-2,12-diethylerythromycin A Et H H Me Me H2,8,10,12-Tetradesmethyl-12-ethylerythromycin A Et H H Me Et Me4,8,10,12-Tetradesmethyl-4,12-diethylerythromycin A Et H H Me H Me4,8,10,12-Tetradesmethyl-12-ethylerythromycin A Et H H Et Me Me6,8,10,12-Tetradesmethyl-6,12-diethylerythromycin A Et H H H Me Me6,8,10,12-Tetradesmethyl-12-ethylerythromycin A Et Et H Me Me Et2,6,10,12-Tetradesmethyl-2,10,12-triethylerythromycin A Et Et H Me Me H2,6,10,12-Tetradesmethyl-10,12-diethylerythromycin A Et Et H Me Et Me4,8,10,12-Tetradesmethyl-4,10,12-triethylerythromycin A Et Et H Me H Me4,8,10,12-Tetradesmethyl-10,12-diethylerythromycin A Et Et H Et Me Me6,8,10,12-Tetradesmethyl-6,10,12-triethylerythromycin A Et Et H H Me Me6,8,10,12-Tetradesmethyl-10,12-diethylerythromycin A Et H Et Me Me Et2,8,10,12-Tetradesmethyl-2,8,12-triethylerythromycin A Et H Et Me Me H2,8,10,12-Tetradesmethyl-8,12-diethylerythromycin A Et H Et Me Et Me4,8,10,12-Tetradesmethyl-4,8,12-triethylerythromycin A Et H Et Me H Me4,8,10,12-Tetradesmethyl-8,12-diethylerythromycin A Et H Et Et Me Me6,8,10,12-Tetradesmethyl-6,8,12-triethylerythromycin A Et H Et H Me Me6,8,10,12-Tetradesmethyl-8,12-diethylerythromycin A Et Et Et Me Me Et2,6,10,12-Tetradesmethyl-2,8,10,12-tetraethylerythromycin A Et Et Et MeMe H 2,6,10,12-Tetradesmethyl-8,10,12-triethylerythromycin A Et Et Et MeEt Me 4,8,10,12-Tetradesmethyl-4,8,10,12-tetraethylerythromycin A Et EtEt Me H Me 4,8,10,12-Tetradesmethyl-8,10,12-triethylerythromycin Et EtEt Et Me Me 6,8,10,12-Tetradesmethyl-6,8,10,12-tetraethylerythromycin AEt Et Et H Me Me 6,8,10,12-Tetradesmethyl-8,10,12-triethylerythromycin HH Me H Me Et 2,6,10,12-Tetradesmethyl-2-ethylerythromycin A H H Me H MeH 2,6,10,12-Tetradesmethylerythromycin A H H Me H Et Me4,6,10,12-Tetradesmethyl-4-ethylerythromycin A H H Me H H Me4,6,10,12-Tetradesmethylerythromycin A H Et Me H Me Et2,6,10,12-Tetradesmethyl-2,10-diethylerythromycin A H Et Me H Me H2,6,10,12-Tetradesmethyl-10-ethylerythromycin A H Et Me H Et Me4,6,10,12-Tetradesmethyl-4,10-diethylerythromycin A H Et Me H H Me4,6,10,12-Tetradesmethyl-10-ethylerythromycin A H H Me Et Me Et2,6,10,12-Tetradesmethyl-2,6-diethylerythromycin A H H Me Et Me H2,6,10,12-Tetradesmethyl-6-ethylerythromycin A H H Me Et Et Me4,6,10,12-Tetradesmethyl-4,6-diethylerythromycin A H H Me Et H Me4,6,10,12-Tetradesmethyl-6-ethylerythromycin A H Et Me Et Me Et2,6,10,12-Tetradesmethyl-2,6,10-triethylerythromycin A H Et Me Et Me H2,6,10,12-Tetradesmethyl-6,10-diethylerythromycin A H Et Me Et Et Me4,6,10,12-Tetradesmethyl-4,6,10-triethylerythromycin A H Et Me Et H Me4,6,10,12-Tetradesmethyl-6,10-diethylerythromycin A Et H Me H Me Et2,6,10,12-Tetradesmethyl-2,12-diethylerythromycin A Et H Me H Me H2,6,10,12-Tetradesmethyl-12-ethylerythromycin A Et H Me H Et Me4,6,10,12-Tetradesmethyl-4,12-diethylerythromycin A Et H Me H H Me4,6,10,12-Tetradesmethyl-12-ethylerythromycin A Et Et Me H Me Et2,6,10,12-Tetradesmethyl-2,10,12-triethylerythromycin A Et Et Me H Me H2,6,10,12-Tetradesmethyl-10,12-diethylerythromycin A Et Et Me H Et Me4,6,10,12-Tetradesmethyl-4,10,12-triethylerythromycin A Et Et Me H H Me4,6,10,12-Tetradesmethyl-10,12-diethylerythromycin A Et H Me Et Me Et2,6,10,12-Tetradesmethyl-2,6,12-triethylerythromycin A Et H Me Et Me H2,6,10,12-Tetradesmethyl-6,12--diethylerythromycin A Et H Me Et Et Me4,6,10,12-Tetradesmethyl-4,6,12-triethylerythromycin A Et H Me Et H Me4,6,10,12-Tetradesmethyl-6,12--diethylerythromycin A Et Et Me Et Me Et2,6,10,12-Tetradesmethyl-2,6,10,12-tetraethylerythromycin A Et Et Me EtMe H 2,6,10,12-Tetradesmethyl-6,10,12-triethylerythromycin A Et Et Me EtEt Me 4,6,10,12-Tetradesmethyl-4,6,10,12-tetraethylerythromycin A Et EtMe Et H Me 4,6,10,12-Tetradesmethyl-6,10,12-triethylerythromycin A H HMe Me H Et 2,4,10,12-Tetradesmethyl-2-ethylerythromycin A H H Me Me H H2,4,10,12-Tetradesmethylerythromycin A H Et Me Me H Et2,4,10,12-Tetradesmethyl-2,10-diethylerythromycin A H Et Me Me H H2,4,10,12-Tetradesmethyl-10-ethylerythromycin A H H Me Me Et Et2,4,10,12-Tetradesmethyl-2,4-diethylerythromycin A H H Me Me Et H2,4,10,12-Tetradesmethyl-4-ethylerythromycin A H Et Me Me Et Et2,4,10,12-Tetradesmethyl-2,4,10-triethylerythromycin A H Et Me Me Et H2,4,10,12-Tetradesmethyl-4,10-diethylerythromycin A Et H Me Me H Et2,4,10,12-Tetradesmethyl-2,12-diethylerythromycin A Et H Me Me H H2,4,10,12-Tetradesmethyl-12-ethylerythromycin A Et Et Me Me H Et2,4,10,12-Tetradesmethyl-2,10,12-triethylerythromycin A Et Et Me Me H H2,4,10,12-Tetradesmethyl-10,12-diethylerythromycin A Et H Me Me Et Et2,4,10,12-Tetradesmethyl-2,4,12-triethylerythromycin A Et H Me Me Et H2,4,10,12-Tetradesmethyl-4,12-diethylerythromycin A Et Et Me Me Et Et2,4,10,12-Tetradesmethyl-2,4,10,12-tetraethylerythromycin A Et Et Me MeEt H 2,4,10,12-Tetradesmethyl-4,10,12-triethylerythromycin A H Me H H MeEt 2,6,8,12-Tetradesmethyl-2-ethylerythromycin A H Me H H Me H2,6,8,12-Tetradesmethylerythromycin A H Me H H Et Me4,6,8,12-Tetradesmethyl-4--ethylerythromycin A H Me H H H Me4,6,8,12-Tetradesmethylerythromycin A H Me Et H Me Et2,6,8,12-Tetradesmethyl-2,8-diethylerythromycin A H Me Et H Me H2,6,8,12-Tetradesmethyl-8--ethylerythromycin A H Me Et H Et Me4,6,8,12-Tetradesmethyl-4,,8-diethylerythromycin A H Me Et H H Me4,6,8,12-Tetradesmethyl-8-ethylerythromycin A H Me H Et Me Et2,6,8,12-Tetradesmethyl-2,6-diethylerythromycin A H Me H Et Me H2,6,8,12-Tetradesmethyl-6-ethylerythromycin A H Me H Et Et Me4,6,8,12-Tetradesmethyl-4,6-diethylerythromycin A H Me H Et H Me4,6,8,12-Tetradesmethyl-6-ethylerythromycin A H Me Et Et Me Et2,6,8,12-Tetradesmethyl-2,6,8-triethyerythromycin A H Me Et Et Me H2,6,8,12-Tetradesmethyl-6,8-diethylerythromycin A H Me Et Et Et Me4,6,8,12-Tetradesmethyl-4,6,8-triethylerythromycin A H Me Et Et H Me4,6,8,12-Tetradesmethyl-6,8-diethylerythromycin A Et Me H H Me Et2,6,8,12-Tetradesmethyl-2,12-diethylerythromycin A Et Me H H Me H2,6,8,12-Tetradesmethyl-12-ethylerythromycin A Et Me H H Et Me4,6,8,12-Tetradesmethyl-4,12-diethylerythromycin A Et Me H H H Me4,6,8,12-Tetradesmethyl-12-ethylerythromycin A Et Me Et H Me Et2,6,8,12-Tetradesmethyl-2,8,12-triethylerythromycin A Et Me Et H Me H2,6,8,12-Tetradesmethyl-8,12-diethylerythromycin A Et Me Et H Et Me4,6,8,12-Tetradesmethyl-4,8,12-triethylerythromycin A Et Me Et H H Me4,6,8,12-Tetradesmethyl-8,12-diethylerythromycin A Et Me H Et Me Et2,6,8,12-Tetradesmethyl-2,6,12-triethylerythromycin A Et Me H Et Me H2,6,8,12-Tetradesmethyl-6,12-diethylerythromycin A Et Me H Et Et Me4,6,8,12-Tetradesmethyl-4,6,12-triethylerythromycin A Et Me H Et H Me4,6,8,12-Tetradesmethyl-6,12-diethylerythromycin A Et Me Et Et Me Et2,6,8,12-Tetradesmethyl-2,6,8,12-tetraethylerythromycin A Et Me Et Et MeH 2,6,8,12-Tetradesmethyl-6,8,12-triethylerythromycin A Et Me Et Et EtMe 4,6,8,12-Tetradesmethyl-4,6,8,12-tetraethylerythromycin A Et Me Et EtH Me 4,6,8,12-Tetradesmethyl-6,8,12-triethylerythromycin A H Me Me H HEt 2,4,6,12-Tetradesmethyl-2-ethylerythromycin A H Me Me H H H2,4,6,12-Tetradesmethylerythromycin A H Me Me Et H Et2,4,6,12-Tetradesmethyl-2,6-diethylerythromycin A H Me Me Et H H2,4,6,12-Tetradesmethyl-6-ethylerythromycin A H Me Me H Et Et2,4,6,12-Tetradesmethyl-2,4-diethylerythromycin A H Me Me H Et H2,4,6,12-Tetradesmethyl-4-ethylerythromycin A H Me Me Et Et Et2,4,6,12-Tetradesmethyl-2,4,6-triethylerythromycin A H Me Me Et Et H2,4,6,12-Tetradesmethyl-4,6-diethylerythromycin A Et Me Me H H Et2,4,6,12-Tetradesmethyl-2,12-diethylerythromycin A Et Me Me H H H2,4,6,12-Tetradesmethyl-12-ethylerythromycin A Et Me Me Et H Et2,4,6,12-Tetradesmethyl-2,6,12-triethylerythromycin A Et Me Me Et H H2,4,6,12-Tetradesmethyl-6,12-diethylerythromycin A Et Me Me H Et Et2,4,6,12-Tetradesmethyl-2,4,12-triethylerythromycin A Et Me Me H Et H2,4,6,12-Tetradesmethyl--diethylerythromycin A Et Me Me Et Et Et2,4,6,12-Tetradesmethyl-2,4,6,12-tetraethylerythromycin A Et Me Me Et EtH 2,4,6,12-Tetradesmethyl-4,6,12-triethylerythromycin A Me H H H Me Et2,6,8,10-Tetradesmethyl-2-ethylerythromycin A Me H H H Me H2,6,8,10-Tetradesmethylerythromycin A Me H H H Et Me4,6,8,10-Tetradesmethyl-4-ethylerythromycin A Me H H H H Me4,6,8,10-Tetradesmethylerythromycin A Me H Et H Me Et2,6,8,10-Tetradesmethyl-2,8-diethylerythromycin A Me H Et H Me H2,6,8,10-Tetradesmethyl-8-ethylerythromycin A Me H Et H Et Me4,6,8,10-Tetradesmethyl-4,8-diethylerythromycin A Me H Et H H Me4,6,8,10-Tetradesmethyl-8-ethylerythromycin A Me H H Et Me Et2,6,8,10-Tetradesmethyl-2,6-diethylerythromycin A Me H H Et Me H2,6,8,10-Tetradesmethyl-6-ethylerythromycin A Me H H Et Et Me4,6,8,10-Tetradesmethyl-4,6-diethylerythromycin A Me H H Et H Me4,6,8,10-Tetradesmethyl-6-ethylerythromycin A Me H Et Et Me Et2,6,8,10-Tetradesmethyl-2,6,8-triethylerythromycin A Me H Et Et Me H2,6,8,10-Tetradesmethyl-6,8-diethylerythromycin A Me H Et Et Et Me4,6,8,10-Tetradesmethyl-4,6,8-triethylerythromycin A Me H Et Et H Me4,6,8,10-Tetradesmethyl-6,8-diethylerythromycin A Me Et H H Me Et2,6,8,10-Tetradesmethyl-2,10-diethylerythromycin A Me Et H H Me H2,6,8,10-Tetradesmethyl-10-ethylerythromycin A Me Et H H Et Me4,6,8,10-Tetradesmethyl-4,10-diethylerythromycin A Me Et H H H Me4,6,8,10-Tetradesmethyl-10-ethylerythromycin A Me Et Et H Me Et2,6,8,10-Tetradesmethyl-2,8,10-triethylerythromycin A Me Et Et H Me H2,6,8,10-Tetradesmethyl-8,10-diethylerythromycin A Me Et Et H Et Me4,6,8,10-Tetradesmethyl-4,8,10-triethylerythromycin A Me Et Et H H Me4,6,8,10-Tetradesmethyl-8,10-diethylerythromycin A Me Et H Et Me Et2,6,8,10-Tetradesmethyl-2,6,10-triethylerythromycin A Me Et H Et Me H2,6,8,10-Tetradesmethyl-6,10-diethylerythromycin A Me Et H Et Et Me4,6,8,10-Tetradesmethyl-4,6,10-triethylerythromycin A Me Et H Et H Me4,6,8,10-Tetradesmethyl-6,10-diethylerythromycin A Me Et Et Et Me Et2,6,8,10-Tetradesmethyl-2,6,8,10-tetraethylerythromycin A Me Et Et Et MeH 2,6,8,10-Tetradesmethyl-6,8,10-triethylerythromycin A Me Et Et Et EtMe 4,6,8,10-Tetradesmethyl-4,6,8,10-tetraethylerythromycin A Me Et Et EtH Me 4,6,8,10-Tetradesmethyl-6,8,10-triethylerythromycin A Me H H Me HEt 2,4,8,10-Tetradesmethyl-2-ethylerythromycin A Me H H Me H H2,4,8,10-Tetradesmethylerythromycin A Me H Et Me H Et2,4,8,10-Tetradesmethyl-2,8-diethylerythromycin A Me H Et Me H H2,4,8,10-Tetradesmethyl-8-ethylerythromycin A Me H H Me Et Et2,4,8,10-Tetradesmethyl-2,4-diethylerythromycin A Me H H Me Et H2,4,8,10-Tetradesmethyl-4-ethylerythromycin A Me H Et Me Et Et2,4,8,10-Tetradesmethyl-2,4,8-triethylerythromycin A Me H Et Me Et H2,4,8,10-Tetradesmethyl-4,8-diethylerythromycin A Me Et H Me H Et2,4,8,10-Tetradesmethyl-2,10-diethylerythromycin A Me Et H Me H H2,4,8,10-Tetradesmethyl-10-ethylerythromycin A Me Et Et Me H Et2,4,8,10-Tetradesmethyl-2,8,10-triethylerythromycin A Me Et Et Me H H2,4,8,10-Tetradesmethyl-8,10-diethylerythromycin A Me Et H Me Et Et2,4,8,10-Tetradesmethyl-2,4,10-triethylerythromycin A Me Et H Me Et H2,4,8,10-Tetradesmethyl-4,10-diethylerythromycin A Me Et Et Me Et Et2,4,8,10-Tetradesmethyl-2,4,8,10-tetraethylerythromycin A Me Et Et Me EtH 2,4,8,10-Tetradesmethyl-4,8,10-triethylerythromycin A Me Me H H H Et2,4,6,8-Tetradesmethyl-2-ethylerythromycin A Me Me H H H H2,4,6,8-Tetradesmethylerythromycin A Me Me H Et H Et2,4,6,8-Tetradesmethyl-2,6,-diethylerythromycin A Me Me H Et H H2,4,6,8-Tetradesmethyl-6-ethylerythromycin A Me Me H H Et Et2,4,6,8-Tetradesmethyl-2,4-diethylerythromycin A Me Me H H Et H2,4,6,8-Tetradesmethyl-4-ethylerythromycin A Me Me H Et Et Et2,4,6,8-Tetradesmethyl-2,4,6-triethylerythromycin A Me Me H Et Et H2,4,6,8-Tetradesmethyl-4,6-diethylerythromycin A Me Me Et H H Et2,4,6,8-Tetradesmethyl-2,8-diethylerythromycin A Me Me Et H H H2,4,6,8-Tetradesmethyl-8-ethylerythromycin A Me Me Et Et H Et2,4,6,8-Tetradesmethyl-2,6,8-triethylerythromycin A Me Me Et Et H H2,4,6,8-Tetradesmethyl-6,8-diethylerythromycin A Me Me Et H Et Et2,4,6,8-Tetradesmethyl-2,4,8-triethylerythromycin A Me Me Et H Et H2,4,6,8-Tetradesmethyl-4,8-diethylerythromycin A Me Me Et Et Et Et2,4,6,8-Tetradesmethyl-2,4,6,8-tetraethylerythromycin A Me Me Et Et Et H2,4,6;8-Tetradesmethyl-4,6,8-triethylerythromycin A E. Five Changes H HH H H Me 4,6,8,10,12-Pentadesmethylerythromycin A Et H H H H Me4,6,8,10,12-Pentadesmethyl-12-ethylerythromycin A H Et H H H Me4,6,8,10,12-Pentadesmethyl-10-ethylerythromycin A H H Et H H Me4,6,8,10,12-Pentadesmethyl-8-ethylerythromycin A H H H Et H Me4,6,8,10,12-Pentadesmethyl-6-ethylerythromycin A H H H H Et Me4,6,8,10,12-Pentadesmethyl-4-ethylerythromycin A Et Et H H H Me4,6,8,10,12-Pentadesmethyl-10,12-diethylerythromycin A Et H Et H H Me4,6,8,10,12-Pentadesmethyl-8,12-diethylerythromycin A Et H H Et H Me4,6,8,10,12-Pentadesmethyl-6,12-diethylerythromycin A Et H H H Et Me4,6,8,10,12-Pentadesmethyl-4,12-diethylerythromycin A H Et Et H H Me4,6,8,10,12-Pentadesmethyl-8,10-diethylerythromycin A H Et H Et H Me4,6,8,10,12-Pentadesmethyl-6,10-diethylerythromycin A H Et H H Et Me4,6,8,10,12-Pentadesmethyl-4,10-diethylerythromycin A H H Et Et H Me4,6,8,10,12-Pentadesmethyl-6,8-diethylerythromycin A H H Et H Et Me4,6,8,10,12-Pentadesmethyl-4,8-diethylerythromycin A H H H Et Et Me4,6,8,10,12-Pentadesmethyl-4,6-diethylerythromycin A Et Et Et H H Me4,6,8,10,12-Pentadesmethyl-8,10,12-triethylerythromycin A Et Et H Et HMe 4,6,8,10,12-Pentadesmethyl-6,10,12-triethylerythromycin A Et Et H HEt Me 4,6,8,10,12-Pentadesmethyl-4,10,12-triethylerythromycin A Et H EtEt H Me 4,6,8,10,12-Pentadesmethyl-6,8,12-triethylerythromycin A Et H EtH Et Me 4,6,8,10,12-Pentadesmethyl-4,8,12-triethylerythromycin A Et H HEt Et Me 4,6,8,10,12-Pentadesmethyl-4,6,12-triethylerythromycin A H EtEt Et H Me 4,6,8,10,12-Pentadesmethyl-6,8,10-triethylerythromycin A H EtEt H Et Me 4,6,8,10,12-Pentadesmethyl-4,8,10-triethylerythromycin A H EtH Et Et Me 4,6,8,10,12-Pentadesmethyl-4,6,10-triethylerythromycin A H HEt Et Et Me 4,6,8,10,12-Pentadesmethyl-4,6,8-triethylerythromycin A EtEt Et Et H Me4,6,8,10,12-Pentadesmethyl-6,8,10,12-tetraethylerythromycin A Et Et Et HEt Me 4,6,8,10,12-Pentadesmethyl-4,8,10,12-tetraethylerythromycin A EtEt H Et Et Me4,6,8,10,12-Pentadesmethyl-4,6,10,12-tetraethylerythromycin A Et H Et EtEt Me 4,6,8,10,12-Pentadesmethyl-4,6,8,12-tetraethylerythromycin A H EtEt Et Et Me 4,6,8,10,12-Pentadesmethyl-4,6,8,10-tetraethylerythromycin AEt Et Et Et Et Me4,6,8,10,12-Pentadesmethyl-4,6,8,10,12-pentaethylerythromycin A H H H HMe H 2,6,8,10,12-Pentadesmethylerythromycin A Et H H H Me H2,6,8,10,12-Pentadesmethyl-12-ethylerythromycin A H Et H H Me H2,6,8,10,12-Pentadesmethyl-10-ethylerythromycin A H H Et H Me H2,6,8,10,12-Pentadesmethyl-8-ethylerythromycin A H H H Et Me H2,6,8,10,12-Pentadesmethyl-6-ethylerythromycin A H H H H Me Et2,6.8,10,12-Pentadesmethyl-2-ethylerythromycin A Et Et H H Me H2,6,8,10,12-Pentadesmethyl-10,12-diethylerythromycin A Et H Et H Me H2,6,8,10,12-Pentadesmethyl-8,12-diethylerythromycin A Et H H Et Me H2,6,8,10,12-Pentadesmethyl-4,12-diethylerythromycin A Et H H H Me Et2,6,8,10,12-Pentadesmethyl-2,12-diethylerythromycin A H Et Et H Me H2,6,8,10,12-Pentadesmethyl-8,10-diethylerythromycin A H Et H Et Me H2,6,8,10,12-Pentadesmethyl-6,10-diethylerythromycin A H Et H H Me Et2,6,8,10,12-Pentadesmethyl-2,10-diethylerythromycin A H H Et Et Me H2,6,8,10,12-Pentadesmethyl-6,8-diethylerythromycin A H H Et H Me Et2,6,8,10,12-Pentadesmethyl-2,8-diethylerythromycin A H H H Et Me Et2,6,8,10,12-Pentadesmethyl-2,6-diethylerythromycin A Et Et Et H Me H2,6,8,10,12-Pentadesmethyl-8,10,12-triethylerythromycin A Et Et H Et MeH 2,6,8,10,12-Pentadesmethyl-6,10,12-triethylerythromycin A Et Et H H MeEt 2,6,8,10,12-Pentadesmethyl-2,10,12-triethylerythromycin A Et H Et EtMe H 2,6,8,10,12-Pentadesmethyl-6,8,12-triethylerythromycin A Et H Et HMe Et 2,6,8,10,12-Pentadesmethyl-2,8,12-triethylerythromycin A Et H H EtMe Et 2,6,8,10,12-Pentadesmethyl-2,6,12-triethylerythromycin A H Et EtEt Me H 2,6,8,10,12-Pentadesmethyl-6,8,10-triethylerythromycin A H Et EtH Me Et 2,6,8,10,12-Pentadesmethyl-2,8,10-triethylerythromycin A H Et HEt Me Et 2,6,8,10,12-Pentadesmethyl-2,6,10-triethylerythromycin A H H EtEt Me Et 2,6,8,10,12-Pentadesmethyl-2,6,8-triethylerythromycin A Et EtEt Et Me H 2,6,8,10,12-Pentadesmethyl-6,8,10,12-tetraethylerythromycin AEt Et Et H Me Et2,6,8,10,12-Pentadesmethyl-2,8,10,12-tetraethylerythromycin A Et Et H EtMe Et 2,6,8,10,12-Pentadesmethyl-2,6,10,12-tetraethylerythromycin A AEtH Et Et Me Et 2,6,8,10,12-Pentadesmethyl-2,6,8,12-tetraethylerythromycinA H Et Et Et Me Et2,6,8,10,12-Pentadesmethyl-2,6,8,10-tetraethylerythromycin A Et Et Et EtMe Et 2,6,8,10,12-Pentadesmethyl-2,6,,8,10,12-pentaethylerythromycin A HH H Me H H 2,4,8,10,12-Pentadesmethylerythromycin A Et H H Me H H2,4,8,10,12-Pentadesmethyl-12-ethylerythromycin A H Et H Me H H2,4,8,10,12-Pentadesmethyl-10-ethylerythromycin A H H Et Me H H2,4,8,10,12-Pentadesmethyl-8-ethylerythromycin A H H H Me Et H2,4,8,10,12-Pentadesmethyl-4-ethylerythromycin A H H H Me H Et2,4,8,10,12-Pentadesmethyl-2-ethylerythromycin A Et Et H Me H H2,4,8,10,12-Pentadesmethyl-10,12-diethylerythromycin A Et H Et Me H H2,4,8,10,12-Pentadesmethyl-8,12-diethylerythromycin A Et H H Me Et H2,4,8,10,12-Pentadesmethyl-4,12-diethylerythromycin A Et H H Me H Et2,4,8,10,12-Pentadesmethyl-2,12-diethylerythromycin A H Et Et Me H H2,4,8,10,12-Pentadesmethyl-8,10-diethylerythromycin A H Et H Me Et H2,4,8,10,12-Pentadesmethyl-4,10-diethylerythromycin A H Et H Me H Et2,4,8,10,12-Pentadesmethyl-2,10-diethylerythromycin A H H Et Me Et H2,4,8,10,12-Pentadesmethyl-4,8-diethylerythromycin A H H Et Me H Et2,4,8,10,12-Pentadesmethyl-2,8-diethylerythromycin A H H H Me Et Et2,4,8,10,12-Pentadesmethyl-2,4-diethylerythromycin A Et Et Et Me H H2,4,8,10,12-Pentadesmethyl-8,10,12-triethylerythromycin A Et Et H Me EtH 2,4,8,10,12-Pentadesmethyl-4,10,12-triethylerythromycin A Et Et H Me HEt 2,4,8,10,12-Pentadesmethyl-2,10,12-triethylerythromycin A Et H Et MeEt H 2,4,8,10,12-Pentadesmethyl-4,8,12-triethylerythromycin A Et H Et MeH Et 2,4,8,10,12-Pentadesmethyl-2,8,12-triethylerythromycin A Et H H MeEt Et 2,4,8,10,12-Pentadesmethyl-2,4,12-triethylerythromycin A H Et EtMe Et H 2,4,8,10,12-Pentadesmethyl-4,8,10-triethylerythromycin A H Et EtMe H Et 2,4,8,10,12-Pentadesmethyl-2,8,10-triethylerythromycin A H Et HMe Et Et 2,4,8,10,12-Pentadesmethyl-2,4,10-triethylerythromycin A H H EtMe Et Et 2,4,8,10,12-Pentadesmethyl-2,4,8-triethylerythromycin A Et EtEt Me Et H 2,4,8,10,12-Pentadesmethyl-4,8,10,12-tetraethylerythromycin AEt Et Et Me H Et2,4,8,10,12-Pentadesmethyl-2,8,10,12-tetraethylerythromycin A Et Et H MeEt Et 2,4,8,10,12-Pentadesmethyl-2,4,10,12-tetraethylerythromycin A Et HEt Me Et Et 2,4,8,10,12-Pentadesmethyl-2,4,8,12-tetraethylerythromycin AH Et Et Me Et Et2,4,8,10,12-Pentadesmethyl-2,4,8,10-tetraethylerythromycin A Et Et Et MeEt Et 2,4,8,10,12-Pentadesmethyl-2,4,8,10,12-pentaethylerytthromycin A HH Me H H H 2,4,6,10,12-Pentadesmethylerythromycin Et H Me H H H2,4,6,10,12-Pentadesmethyl-12-ethylerythromycin A H Et Me H H H2,4,6,10,12-Pentadesmethyl-10-ethylerythromycin A H H Me Et H H2,4,6,10,12-Pentadesmethyl-6-ethylerythromycin A H H Me H Et H2,4,6,10,12-Pentadesmethyl-4-ethylerythromycin A H H Me H H Et2,4,6,10,12-Pentadesmethyl-2-ethylerythromycin A Et Et Me H H H2,4,6,10,12-Pentadesmethyl-10,12-diethylerythromycin A Et H Me Et H H2,4,6,10,12-Pentadesmethyl-6,12-diethylerythromycin A Et H Me H Et H2,4,6,10,12-Pentadesmethyl-4,12-diethylerythromycin A Et H Me H H Et2,4,6,10,12-Pentadesmethyl-2,12-diethylerythromycin A H Et Me Et H H2,4,6,10,12-Pentadesmethyl-6,10-diethylerythromycin A H Et Me H Et H2,4,6,10,12-Pentadesmethyl-4,10-diethylerythromycin A H Et Me H H Et2,4,6,10,12-Pentadesmethyl-2,10-diethylerythromycin A H H Me Et Et H2,4,6,10,12-Pentadesmethyl-4,6-diethylerythromycin A H H Me Et H Et2,4,6,10,12-Pentadesmethyl-2,6-diethylerythromycin A H H Me H Et Et2,4,6,10,12-Pentadesmethyl-2,4-diethylerythromycin A Et Et Me Et H H2,4,6,10,12-Pentadesmethyl-6,10,12-triethylerythromycin A Et Et Me H EtH 2,4,6,10,12-Pentadesmethyl-4,10,12-triethylerythromycin A Et Et Me H HEt 2,4,6,10,12-Pentadesmethyl-2,10,12-triethylerythromycin A Et H Me EtEt H 2,4,6,10,12-Pentadesmethyl-4,6,12-triethylerythromycin A Et H Me EtH Et 2,4,6,10,12-Pentadesmethyl-2,6,12-triethylerythromycin A Et H Me HEt Et 2,4,6,10,12-Pentadesmethyl-2,4,12-triethylerythromycin A H Et MeEt Et H 2,4,6,10,12-Pentadesmethyl-4,6,10-triethylerythromycin A H Et MeEt H Et 2,4,6,10,12-Pentadesmethyl-2,6,10-triethylerythromycin A H Et MeH Et Et 2,4,6,10,12-Pentadesmethyl-2,4,10-triethylerythromycin A H H MeEt Et Et 2,4,6,10,12-Pentadesmethyl-2,4,6-triethylerythromycin A Et EtMe Et Et H 2,4,6,10,12-Pentadesmethyl-4,6,10,12-tetraethylerythromycin AEt Et Me Et H Et2,4,6,10,12-Pentadesmethyl-2,6,10,12-tetraethylerythromycin A Et Et Me HEt Et 2,4,6,10,12-Pentadesmethyl-2,4,10,12-tetraethylerythromycin A Et HMe Et Et Et 2,4,6,10,12-Pentadesmethyl-2,4,6,12-tetraethylerythromycin AH Et Me Et Et Et2,4,6,10,12-Pentadesmethyl-2,4,6,10-tetraethylerythromycin A Et Et Me EtEt Et 2,4,6,10,12-Pentadesmethyl-2,4,6,10,12-pentaethylerythromycin A HMe H H H H 2,4,6,8,12-Pentadesmethylerythromycin A Et Me H H H H2,4,6,8,12-Pentadesmethyl-12-ethylerythromycin A H Me Et H H H2,4,6,8,12-Pentadesmethyl-8-ethylerythromycin A H Me H Et H H2,4,6,8,12-Pentadesmethyl-6-ethylerythromycin A H Me H H Et H2,4,6,8,12-Pentadesmethyl-4-ethylerythromycin A H Me H H H Et2,4,6,8,12-Pentadesmethyl-2-ethylerythromycin A Et Me Et H H H2,4,6,8,12-Pentadesmethyl-8,12-diethylerythromycin A Et Me H Et H H2,4,6,8,12-Pentadesmethyl-6,12-diethylerythromycin A Et Me H H Et H2,4,6,8,12-Pentadesmethyl-4,12-diethylerythromycin A Et Me H H H Et2,4,6,8,12-Pentadesmethyl-2,12-diethylerythromycin A H Me Et Et H H2,4,6,8,12-Pentadesmethyl-6,8-diethylerythromycin A H Me Et H Et H2,4,6,8,12-Pentadesmethyl-4,8-diethylerythromycin A H Me Et H H Et2,4,6,8,12-Pentadesmethyl-2,8-diethylerythromycin A H Me H Et Et H2,4,6,8,12-Pentadesmethyl-4,6-diethylerythromycin A H Me H Et H Et2,4,6,8,12-Pentadesmethyl-2,6-diethylerythromycin A H Me H H Et Et2,4,6,8,12-Pentadesmethyl-2,4-diethylerythromycin A Et Me Et Et H H2,4,6,8,12-Pentadesmethyl-6,8,12-triethylerythromycin A Et Me Et H Et H2,4,6,8,12-Pentadesmethyl-4,8,12-triethylerythromycin A Et Me Et H H Et2,4,6,8,12-Pentadesmethyl-2,8,12-triethylerythromycin A Et Me H Et Et H2,4,6,8,12-Pentadesmethyl-4,6,12-triethylerythromycin A Et Me H Et H Et2,4,6,8,12-Pentadesmethyl-2,6,12-triethylerythromycin A Et Me H H Et Et2,4,6,8,12-Pentadesmethyl-2,4,12-triethylerythromycin A H Me Et Et Et H2,4,6,8,12-Pentadesmethyl-4,6,8-triethylerythromycin A H Me Et Et H Et2,4,6,8,12-Pentadesmethyl-2,6,8-triethylerythromycin A H Me Et H Et Et2,4,6,8,12-Pentadesmethyl-2,4,8-triethylerythromycin A H Me H Et Et Et2,4,6,8,12-Pentadesmethyl-2,4,6-triethylerythromycin A Et Me Et Et Et H2,4,6,8,12-Pentadesmethyl-4,6,8-triethylerythromycin A Et Me Et Et H Et2,4,6,8,12-Pentadesmethyl-2,6,8,12-tetraethylerythromycin A Et Me Et HEt Et 2,4,6,8,12-Pentadesmethyl-2,4,8,12-tetraethylerythromycin A Et MeH Et Et Et 2,4,6,8,12-Pentadesmethyl-2,4,6,12-tetraethylerythromycin A HMe Et Et Et Et 2,4,6,8,12-Pentadesmethyl-2,4,6,8-tetraethylerythromycinA Et Me Et Et Et Et2,4,6,8,12-Pentadesmethyl-2,4,6,8,12-pentaethylerythromycin A Me H H H HH 2,4,6,8,10-Pentadesmethylerythromycin A Me Et H H H H2,4,6,8,10-Pentadesmethyl-10-ethylerythromycin A Me H Et H H H2,4,6,8,10-Pentadesmethyl-8-ethylerythromycin A Me H H Et H H2,4,6,8,10-Pentadesmethyl-6-ethylerythromycin A Me H H H Et H2,4,6,8,10-Pentadesmethyl-4-ethylerythromycin A Me H H H H Et2,4,6,8,10-Pentadesmethyl-2-ethylerythromycin A Me Et Et H H H2,4,6,8,10-Pentadesmethyl-8,10 diethylerythromycin A Me Et H Et H H2,4,6,8,10-Pentadesmethyl-6,10 diethylerythromycin A Me Et H H Et H2,4,6,8,10-Pentadesmethyl-4,10 diethylerythromycin A Me Et H H H Et2,4,6,8,10-Pentadesmethyl-2,10 diethylerythromycin A Me H Et Et H H2,4,6,8,10-Pentadesmethyl-6,8-diethylerythromycin A Me H Et H Et H2,4,6,8,10-Pentadesmethyl-4,8-diethylerythromycin A Me H Et H H Et2,4,6,8,10-Pentadesmethyl-2,8-diethylerythromycin A Me H H Et Et H2,4,6,8,10-Pentadesmethyl-4,6-diethylerythromycin A Me H H Et H Et2,4,6,8,10-Pentadesmethyl-2,6-diethylerythromycin A Me H H H Et Et2,4,6,8,10-Pentadesmethyl-2,4-diethylerythromycin A Me Et Et Et H H2,4,6,8,10-Pentadesmethyl-6,8,10-triethylerythromycin A Me Et Et H Et H2,4,6,8,10-Pentadesmethyl-4,8,10-triethylerythromycin A Me Et Et H H Et2,4,6,8,10-Pentadesmethyl-2,8,10-triethylerythromycin A Me Et H Et Et H2,4,6,8,10-Pentadesmethyl-4,6,10-triethylerythromycin A Me Et H Et H Et2,4,6,8,10-Pentadesmethyl-2,6,10-triethylerythromycin A Me Et H H Et Et2,4,6,8,10-Pentadesmethyl-2,4,10-triethylerythromycin A Me H Et Et Et H2,4,6,8,10-Pentadesmethyl-4,6,8-triethylerythromycin A Me H Et Et H Et2,4,6,8,10-Pentadesmethyl-2,6,8-triethylerythromycin A Me H Et H Et Et2,4,6,8,10-Pentadesmethyl-2,4,8-triethylerythromycin A Me H H Et Et Et2,4,6,8,10-Pentadesmethyl-2,4,6-triethylerythromycin A Me Et Et Et Et H2,4,6,8,10-Pentadesmethyl-4,6,8,10-tetraethylerythromycin A Me Et Et EtH Et 2,4,6,8,10-Pentadesmethyl-2,6,8,10-tetraethylerythromycin A Me EtEt H Et Et 2,4,6,8,10-Pentadesmethyl-2,4,8,10-tetraethylerythromycin AMe Et H Et Et Et2,4,6,8,10-Pentadesmethyl-2,4,6,10-tetraethylerythromycin A Me H Et EtEt Et 2,4,6,8,10-Pentadesmethyl-2,4,6,8-tetraethylerythromycin A Me EtEt Et Et Et 2,4,6,8,10-Pentadesmethyl-2,4,6,8,10-pentaethylerythromycinA F. Six Changes H H H H H H 2,4,6,8,10,12-Hexadesmethylerythromycin AEt H H H H H 2,4,6,8,10,12-Hexadesmethyl-12-ethylerythromycin A H Et H HH H 2,4,6,8,10,12-Hexadesmethyl-10-ethylerythromycin A H H Et H H H2,4,6,8,10,12-Hexadesmethyl-8-ethylerythromycin A H H H Et H H2,4,6,8,10,12-Hexadesmethyl-6-ethylerythromycin A H H H H Et H2,4,6,8,10,12-Hexadesmethyl-4-ethylerythromycin A H H H H H Et2,4,6,8,10,12-Hexadesmethyl-2-ethylerythromycin A Et Et H H H H2,4,6,8,10,12-Hexadesmethyl-10,12-diethylerythromycin A Et H Et H H H2,4,6,8,10,12-Hexadesmethyl-8,12-diethylerythromycin A Et H H Et H H2,4,6,8,10,12-Hexadesmethyl-6,12-diethylerythromycin A Et H H H Et H2,4,6,8,10,12-Hexadesmethyl-4,12-diethylerythromycin A Et H H H H Et2,4,6,8,10,12-Hexadesmethyl-2,12-diethylerythromycin A H Et Et H H H2,4,6,8,10,12-Hexadesmethyl-8,10-diethylerythromycin A H Et H Et H H2,4,6,8,10,12-Hexadesmethyl-6,10-diethylerythromycin A H Et H H Et H2,4,6,8,10,12-Hexadesmethyl-4,10-diethylerythromycin A H Et H H H Et2,4,6,8,10,12-Hexadesmethyl-2,10-diethylerythromycin A H H Et Et H H2,4,6,8,10,12-Hexadesmethyl-6,8-diethylerythromycin A H H Et H Et H2,4,6,8,10,12-Hexadesmethyl-4,8-diethylerythromycin A H H Et H H Et2,4,6,8,10,12-Hexadesmethyl-2,8-diethylerythromycin A H H H Et Et H2,4,6,8,10,12-Hexadesmethyl-4,6-diethylerythromycin A H H H Et H Et2,4,6,8,10,12-Hexadesmethyl-2,6-diethylerythromycin A H H H H Et Et2,4,6,8,10,12-Hexadesmethyl-2,4-diethylerythromycin A Et Et Et H H H2,4,6,8,10,12-Hexadesmethyl-8,10,12-triethylerythromycin A Et Et H Et HH 2,4,6,8,10,12-Hexadesmethyl-6,10,12-triethylerythromycin A Et Et H HEt H 2,4,6,8,10,12-Hexadesmethyl-4,10,12-triethylerythromycin A Et Et HH H Et 2,4,6,8,10,12-Hexadesmethyl-2,10,12-triethylerythromycin A Et HEt Et H H 2,4,6,8,10,12-Hexadesmethyl-6,8,12-triethylerythromycin A Et HEt H Et H 2,4,6,8,10,12-Hexadesmethyl-4,8,12-triethylerythromycin A Et HEt H H Et 2,4,6,8,10,12-Hexadesmethyl-2,8,12-triethylerythromycin A Et HH Et Et H 2,4,6,8,10,12-Hexadesmethyl-4,6,12-triethylerythromycin A Et HH Et H Et 2,4,6,8,10,12-Hexadesmethyl-2,6,12-triethylerythromycin A Et HH H Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,12-triethylerythromycin A H EtEt Et H H 2,4,6,8,10,12-Hexadesmethyl-6,8,10-triethylerythromycin A H EtEt H Et H 2,4,6,8,10,12-Hexadesmethyl-4,8,10-triethylerythromycin A H EtEt H H Et 2,4,6,8,10,12-Hexadesmethyl-2,8,10-triethylerythromycin A H EtH Et Et H 2,4,6,8,10,12-Hexadesmethyl-4,6,10-triethylerythromycin A H EtH Et H Et 2,4,6,8,10,12-Hexadesmethyl-2,6,10-triethylerythromycin A H EtH H Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,10-triethylerythromycin A H HEt Et Et H 2,4,6,8,10,12-Hexadesmethyl-4,6,8-triethylerythromycin A H HEt Et H Et 2,4,6,8,10,12-Hexadesmethyl-2,6,8-triethylerythromycin A H HEt H Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,8-triethylerythromycin A H HH Et Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,6-triethylerythromycin A EtEt Et Et H H2,4,6,8,10,12-Hexadesmethyl-6,8,10,12-tetraethylerythromycin A Et Et EtH Et H 2,4,6,8,10,12-Hexadesmethyl-4,8,10,12-tetraethylerythromycin A EtEt Et H H Et2,4,6,8,10,12-Hexadesmethyl-2,8,10,12-tetraethylerythromycin A Et Et HEt Et H 2,4,6,8,10,12-Hexadesmethyl-4,6,10,12-tetraethylerythromycin AEt Et H Et H Et2,4,6,8,10,12-Hexadesmethyl-2,6,10,12-tetraethylerythromycin A Et Et H HEt Et 2,4,6,8,10,12-Hexadesmethyl-2,4,10,12-tetraethylerythromycin A EtH Et Et Et H 2,4,6,8,10,12-Hexadesmethyl-4,6,8,12-tetraethylerythromycinA Et H Et Et H Et2,4,6,8,10,12-Hexadesmethyl-2,6,8,12-tetraethylerythromycin A Et H Et HEt Et 2,4,6,8,10,12-Hexadesmethyl-2,4,8,12-tetraethylerythromycin A Et HH Et Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,6,12-tetraethylerythromycin AH Et Et Et Et H2,4,6,8,10,12-Hexadesmethyl-4,6,8,10-tetraethylerythromycin A H Et Et EtH Et 2,4,6,8,10,12-Hexadesmethyl-2,6,8,10-tetraethylerythromycin A H EtEt H Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,8,10-tetraethylerythromycin AH Et H Et Et Et2,4,6,8,10,12-Hexadesmethyl-2,4,6,10-tetraethylerythromycin A H H Et EtEt Et 2,4,6,8,10,12-Hexadesmethyl-2,4,6,8-tetraethylerythromycin A Et EtEt Et Et H2,4,6,8,10,12-Hexadesmethyl-4,6,8,10,12-pentaethylerythromycin A Et EtEt Et H Et2,4,6,8,10,12-Hexadesmethyl-2,6,8,10,12-pentaethylerythromycin A Et EtEt H Et Et2,4,6,8,10,12-Hexadesmethyl-2,4,8,10,12-pentaethylerythromycin A Et Et HEt Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,6,10,12-pentaethylerythromycinA Et H Et Et Et Et2,4,6,8,10,12-Hexadesmethyl-2,4,6,,8,12-pentaethylerythromycin A H Et EtEt Et Et 2,4,6,8,10,12-Hexadesmethyl-2,4,6,8,10-pentaethylerythromycin AEt Et Et Et Et Et2,4,6,8,10,12-Hexadesmethyl-2,4,6,8,10,12-hexaethylerythromycin

[0128] Although in the Examples that follow the AT-encoding DNAfragments from S. hygroscopicus ATCC 29253, S. venezuelae ATCC 15439, S.caelestis NRRL-2821 were used to replace resident AT-encoding DNAfragments in the eryPKS to yield desmethyl, desmethylethyl, anddesmethylhydroxyerythromycins, it is understood that many malonate,ethylmalonate, and hydroxymalonate AT-encoding DNA fragments can be usedin place of or in addition to the heterologous malonate, ethylmalonate,and hydroxymalonate-At DNA fragments described herein to produce thesame desmethyl, desmethylethyl, and desmethylhydroxyerythromycincompounds. Example of DNA fragments encoding malonate-AT domains thatcan be used in place of or in addition to those specifically descirbedin the Examples below include but are not limited to the DNA fragmentsencoding AT domains from modules 2, 5, 8, 9, 11, or 12 of the rapamycinPKS genes from S. hygroscopicus, the At domain from module 2 of the PKSresponsible the synthesis of methymycin or pikromycin by S. venezuelae,the AT domains from modules 3 and 7 of the PKS responsible for thesynthesis of tylosin by S. fradiae, or the AT domains from modules 1, 2,3 and 7 of the PKS responsible for the synthesis of spiramycin by S.ambofaciens. Examples of DNA fragments encoding ethylmalonate-AT domainsthat can be used in place of or in addition to those specificallydescribed in the Examples below include but are not limited to the DNAfragments encoding the AT domain from module 5 of the spiramycin PKSgenes from S. ambofaciens, the AT domain from module 5 of themaridomycin PKS genes of S. hygroscopicus. Examples of DNA fragmentsencoding hydroxymalonate-AT domains that can be used in place of or inaddition to those specifically described in the Examples below includebut are not limited to the DNA fragments encoding the AT domain frommodule 6 of the spiramycin PKS genes from S. ambofaciens, the AT domainfrom module 6 of the maridomycin PKS genes from S. hygroscpicus, and theAT domain from module 6 of the leucomycin PKS genes fromStreptoverticillium kitasatoensis. Thus the use of any and all DNAfragments encoding malonate, ethylmalonate, and hydroxymalonate-ATs toreplace any of the resident DNA fragments encoding methylmalonate-ATs inthe eryPKS genes to result in the production of novel derivatives oferythromycin; are considered within the scope of the present invention.

[0129] Furthermore, those of ordinary skill understand that followingthe methods described herein for replacement of resident AT-encoding DNAfragments in the eryPKS, the DNA fragments encoding malonate-ATs in S.hygroscopicus, S. venezuelae, or S. caelestis, and ethylmalonate orhydroxymalonate-ATs in S. caelestis may be replace with thoseAT-encoding DNA fragments from the eryPKS which utilize methylmalonylCoA as a substrate.

[0130] As with the eryPKS, all combinations are contemplated, leading tothe production of, for example, 13-methylrapamycin, 15-methylrapamycin,33-methylrapamycin, 13,15-dimethylrapamycin,13,15,33-trimethylrapamycin, and 10-methylpikromycin.

[0131] The methods of the present invention are widlely applicable toall erythromycin-producing microorganisms, of which a non-exhaustivelist includes Saccharopolyspora species, Streptomyces griseoplanus,Nocardia sp., Micromonospora sp., Arthrobacter sp. and Streptomycesantibioticus. Of these, Sac. erythlaea is the most preferred. Otherhosts, which normally do not produce erythromycin but into which theerythromycin biosynthesis genes call be introduced by cloning, call alsobe employd. Such strains include but are not limited to Streptomycescoelicolor and Streptomyces lividans or Bacillus subtilis, as examples.In each of the other erythromycin-producing strains, replacement of theresident At domains in the erythromycin PKS is conducted by doublehomologous recombination using cloned eryPKS sequences on both sides ofthe At domain to be replaced to effect the switching of the resident Atwith a heterologous AT as illustrated in the Examples that follow.

[0132] Many other variations of the methods that are illustrated in theExamples that follow will occur to those skilled in the art. Forexample, whereas the plasmids pUC18, pUC19, pGEM3Zf, and pCS5 wereemployed in the present invention for the cloning of the LigAT2, venAT,rapAT14, NidAT5, or NidAT6-encoding DNA fragments and construction ofthe integration vectors, other plasmids, phage, or phagemids includingbut not limited to pBR322, pACYC184, M31mp18, M13mp19, pGEM7Zf and thelike can be used in their place to allow the same constructions to bemade. Furthermore, many alternative strategies can be followed for thecloning of the heterologous AT-encoding DNA fragments into integrationvectors that enable homologous recombination to occur in correspondingregions of the eryPKS. Examples of alternative strategies include theuse of longer or shorter fragments of DNA corresponding to either the ATdomains or the flanking sequences, using different restriction sites forthe cloning of the AT domains or the adjacent flanking sequences, orchanging the sequence of a resident AT-encoding DNA fragment so that itexpresses a domain which recognizes malonyl CoA as a substrate ratherthan methylmalonyl CoA. All such variations are within the scope of thepresent invention. Similarly, employing alternative strategies tointroduce DNA into Sac. erythraea or other erythromycin-producing hostsfor the purpose of effecting gene exchange to result in the productionof novel erythromycins, such as conjugation, transduction orelectroporation are also included within the scope of the presentinvention.

[0133] Those skilled in the art also understand that erythromycins B, Cand D are naturally occurring forms of erythromycin and therefore wouldbe produced as novel derivatives in Sac. erythraea by the modificationsdisclosed herein. Production of these forms may be further enhanced byinactivation of eryK (Stassi, D. et al. J. Bacteriology, 175:182-189,(1993)) to yield erythromycin B derivatives, eryG (S. F. Haydock et al.Mol. Gen. Genet. 230:120-128(1991)) to yield erythromycin C derivativesand eryK and eryG to yield erythromycin D derivatives. Furthermore, inSac. erythraea, 6-deoxy forms of the novel erythromycins A, B, C and Dcan be generated by inactivation of eryF (J. M. Weber et al. Science252:114-117(1991)) (in addition to those specified above), which encodesthe hydroxylase responsible for hydroxylating the C-6 position. Inaddition, conversion ol 6-deoxy forms of the novel erythromnycins A, B,C and D to their corresponding erythromycin A, B, C, and D derivativesmay be accomplished by cloning additional copies or by employing othermeans of overexpression of the eryf gene in the production host.Similarly, conversion of novel forms of erythromycins B, C and D tonovel forms of erythromycin A may be achieved by expressing oroverexpressing eryK and/or eryG in the production host. Themethodologies for generating erythromycinis B, C and D and6-deoxyerythromycins A, B, C and D are well known to those of ordinaryskill in the art.

[0134] Those skilled in the art also understand that erythronolide B and3-α-mycarosylerythronolide B are naturally occurring intermediates inthe biosynthesis of erythromycin and therefore would be produced asnovel intermediates in Sac. erythraea by the modifications disclosedherein. Production of these forms may be further enhanced byinactivation of any of the eryB genes to yield erythronolide B or eryCgenes to yield 3-α-L-mycarosylerythronolide B (Weber et al. J.Bacteriol.172:2372-2383 (1990)) and Haydock et al. Mol. Gen. Genet.230:120-128 (1991)). Furthermore, 6-deoxy forms of these novelintermediates can be generated by inactivation of eryF as describedabove. The methodologies for generating erythronolide B and3-α-mycarosylerythronolide B, as well as their 6-deoxy derivatives, arewell known to those of ordinary skill in the art.

Bacterial Strains, Plasmid Vectors, and Growth Media

[0135] The erythromycin-producing microorganism used to practice thefollowing examples of the invention was Sac. erythraea ER720 (J. P.Dewitt, J. Bacteriol. 164:969 (1985)). The host strain for the growth ofE. coli derived plasmids was D115α from GIBCO BRL, Gaitherburg, md.).The S. hygroscopicus strain that carries the Lig-PKS cluster isavailable from the American Type Culture Collection, Bethesa, Md. underthe accession number ATCC 29253. The S. venezuelae strain that carriesthe venAT domain described herein is available from the American TypeCulture Collection, Bethesda, Md. under the accession number ATCC 15439.

[0136]E. coli bacteria carrying pUC18/venAT has been deposited at theAgricultural Research Culture Collection (NRRL), 1815 N. UniversityStreet, Peoria, Ill. 61604 U.S.A., as of Dec. 23, 1996, under the termsof the Budapest Treaty and will be maintained for a period of thirty(30) years from the (late of deposit, or for five (5) years after thelast request for the deposit, or for the enforceable period of the U.S.patent, whichever is longer. The deposit and any other depositedmaterial described herein are provided for convenience only, and are notrequired to practice the present invention in view of the teachingsprovided herein. The DNA sequence in all of the deposited material isincorporated herein by reference. E. coli bacteria carrying pUC18/venATwas accorded NRRL Deposit No B-21652.

[0137] Plasmid pUC18 and pUC19 can be obtained from GIBCO BRL. PlasmidpCS5, a multifunctional vector for integrative transformation of Sac.erythraea is described in Vara, et al., J. Bacteriology, 171:5872-5881(1989) and is referred to therein as pWHM3. Cosmid pNJ1 is described inTuan, et al., Gene, 90: 21-29 (1990).

[0138]Sac. erythraea was grown for protoplast formation and routineliquid culture in 50 mL of SGGP medium (Yamamoto, et al., J. Antibiotic.39:1304 (1986)), supplemented with 10 μg of thiostrepton/mL for plasmidselection where appropriate.

Reagents and General Methods

[0139] Commercially available reagents were used to make compounds,plasmids and genetic variants of the present invention, includingbutyric acid, ampicillin, thiostrepton, restriction endonucleases,T4-DNA ligase, and calf intestine alkaline phosphatase. The nucleotidesequence of the eryA genes from Sac. erythraea has been deposited in theGenBank database under the accession numbers M63676 and M63677 and arepublicly available.

[0140] Standard molecular biology procedures (Maniatis et al., supra)were used for the construction and characterization of replacementplasmids. Plasmid DNA was routinely isolated by the alkaline lysismethod (H. C. Birnboim and J. Doly, 1979 Nucleic Acids Res. 7:1513) orwith QlAprep Spin Plasmid kit (Qiageni, Inc., Chatsworth, Calif.)according to the manufacturers instructions. Restriction fragments wererecovered from 0.8-1% agarose gels with Prep-A-Gene (BioRad). Theproducts of ligation for each step of the plasmid constructions wereused to transform the intermediate host, E. coli DH5α (GIBCO BRL), whichwas cultured in the presence of ampicillin to select for host cellscarrying recombinant plasmids. Selection for insert DNA with X-gal wasused where appropriate. Typically, LB plates contain 30 mL of LB agar(Maniatis et al., supra). Plasmid DNAs were isolated from individualtransformants that had been grown in liquid culture and characterizedwith respect to known restriction sites. DNA sequence determination wasby cycle sequencing (fmol DNA Sequencing System, Promega Corp. Madison,Wis.) according to the manufacturer's instructions.

[0141] SCM medium consist of 20 g Soytone, 15 g Soluble Starch, 10.5 gMOPS, 1.5 g Yeast Extract and 0.1 g CaCl₂ per liter of distilled H₂O .SGGP medium is described in Yamamoto, et al., 1986, J. Antibiotic.39:1304. PM buffer (per liter) is 200 g sucrose, 0.25 g K₂SO₄ in 890 mLH₂O, with the addition after sterilization of 100 mL 0.25 M TES, pH 7.2,2 mL trace elements solution (Hopwood, et al., 1985, GeneticManipulation of Streptomyces A Laboratory Manual, The John InnesFoundation), 0.08 mL 2.5 M CaCl₂, 10 mL 0.5% KH₂PO₄, 2 mL 2.5 M MgCl₂.

[0142] Integrative transformation of Sac. erythraea protoplasts, androutine growth and sporulation were carried out according to proceduresdescribed in Donadio, et al., 1991, Science 115:97; Weber and Losick,1988, Gene 68:173; and Yamamoto, et al., 1986, J. Antibiotic. 39:1304.

[0143] Oligo primers used in the PCR amplifications and described in theExamples below are as follows: 5′-ATCTACACSTCSGGCACSACSGGCAAGCCSAAGGG-3′SEQ ID NO:3 5′-CTSAAGGCSGGCGGCGCSTACGTSCCSATCGACCC)-3′ SEQ ID NO:45′-CGCGAATTCCTAGGCTGGCGGTGATGTTCA-3′ SEQ ID NO:55′-GCCGGATCCATGCATACGTCGGCAGGGAGGTAC-3′ SEQ ID NO:65′-GCTCGAATTCGCTGGTCGCGGTGCACCT-3′ SEQ ID NO:75′-GACGGATCCGGCCCTAGGCTGCGCCCGGCTCG-3′ SEQ ID NO:85′-TTGGGATCCTATGCATTCCAGCGCGAGCGC-3′ SEQ ID NO:95′-GAGAAGCTTGGCGCGACTTGCCCGCT-3′ SEQ ID NO:105′-TTTTTTAAGCTTGGTACCTGCTCACCGGCAACACCG-3′ SEQ ID NO:115′-TTTTTTGGATCCCTGCAGCCTAGGGTCGGAGGCACTGCCGGT-3′ SEQ ID NO:125′-TTTTTTCTGCAGTATGCATTCCAGGGCAAGCGGTTCT-3′ SEQ ID NO:135′-TTTTTTGAATTCACGCGTTGCCCGCGGCGTAGGCGC-3′ SEQ ID NO:145′-GATCGAATTCCCTAGGACGGCAGTCCTGCTCACC-3′ SEQ ID NO:155′-GATCGGATCCATGCATACGTCGGAAGGTCGACCCG-3′ SEQ ID NO:165′-TTCGAAGAATTCCCTAGGGTTGCCTTCCTGTTCGAC-3′ SEQ ID NO:175′-TTCGAAAAGCTTATGCATAGACCGGCAGATCCACCG-3′ SEQ ID NO:185′-CGGTSAAGTCSAACATCGG-3′ SEQ ID NO:19 5′-GCRATCTCRCCCTGCGARTG-3′ SEQ IDNO:20 5′-GAGAGAGGAACCAACGCGCACGTGATCGTCGAAGAGGCACCAGC-3′ SEQ ID NO:215′-GAGAGAGGATCCGACCTAGGCGCGGAGGTCACCGGCGCGACGGCG-3′ SEQ ID NO:225′-GAGAGACCTAGGAAGCCGGTGTTCGTGTTCCCCGGCCAGGGCT-3′ SEQ ID NO:235′-GAGAGAGGATCCGAGGCCGGCCGTGCGCCCGGACCGAAGACCGCCTC-3′ SEQ ID NO:245′-GAGAGAATTCCCTAGGGTCGCCTTCGTCTTTCCCGGGCAGG-3′ SEQ ID NO:255′-TTGAGATCTTATGCATACGAGGGAAGCGGCACCCTGC-3′ SEQ ID NO:26

[0144] Mass spectrometry was routinely performed with a Finnigan-MAT7000 mass spectrometer etquipped with an atmospheric pressure chemicalionization source (APCl). Electrospray mass spectrometry (ESI-MS) witsperfonred with a Finnigan-MAT 752-7000 mass spectrometer equipped with aFinnigan atmospheric pressure ionization (APl) source. HPLC separationwas carried out on a Hewlett-Packardt 1050 liquid chromatograph using aProdigy ODS (2) column (5 μm, 50×2 mm ) and a gradient elution of 5 mMammonium acetate and methanaol. The flow rate was 0.3 mL/min.

[0145] For large scale preparation of erythromycin derivatives,fermentation beers are typically adjusted to pH 9 with NH₄OH and thenextracted two times with an equal volume of CH₂Cl₂. The pooled extractis then concentrated to a wet oil (approx. 1 g per liter of fermentationbeer). Concentrated extracts are digested in methanol andchromatographed over a column of Sephadex® LH-20 (Pharmacia Biotech,Uppsala, Sweden) in the same solvent. Fractions are tested forbioactivity against Staphylococcus aureus, and active fractions arecombined and concentrated. When additional column chromatography isdesired to reduce sample weight, the concentrated sample is digested ina solvent system consisting of n-heptane, chloroform, ethanol (10:10:1,v/v/v) and chromatographed over a column of Sephadex® LH-20 in the samesystem. Fractions are then analyzed by ¹H NMR, focusing on thecharacteristic erythromycin resonances around δ=5.0 (H-13), δ=4.9(H-1″), and δ=4.4 (H-1′) (Everett and Tyler, J. Chem. Soc. Perkin Trans.1, pg. 2599 (1985)) and pooled according to purity. Alternatively,column chromatography is replaced with an extraction sequence. In thiscase, the initial pooled CH₂Cl₂ extract is concentrated to approximately400 mL. This is extracted twice with equal volumes of 0.05 M aqueouspotassium phosphate with the pH chosen between pH 4.5-6. The aqueousphase is then pooled, adjusted to pH 8-9, and extracted twice with equalvolumes of ethyl acetate. Finally, the ethyl acetate extracts are pooledand concentrated. When additional reduction in sample weight is desired,the extraction sequence is repeated on a 10-50 fold smaller scale,typically yielding about 500 mgs of partially pure material.

[0146] High resolution separation of erythromycin derivatives isobtained by one or more rounds of countercurrent chromatography(Hostettman and Marston, Anal. Chim. Acta, 236:63-76 (1990)). When theweight of the partially pure sample from column chromatography or theextraction sequence is less than 5 g, but greater than 0.5 g, it isdigested in 7 mnL of the upper phase of a solvent system (3:7:5, v/v/v)consisting of n-hexane, ethyl acetate, 0.02 M aqueous potassiumphosphate, with a pH chosen between 6.5-8.0 , and chromatographed on acustom droplet countercurrent chromatography (DCCC) instrument [110vertical columns, 0.4 cm dia.×24 cm length; Hostettmann and Marston,Anal. Chim. Acta, 236:63-76 (1990)] in the same system with the upperphase as the mobile phase. Flow rates of approximately 120-200 mL/hr areemployed. As before, fractions are analyzed by NMR and bioactivity, andpooled according to purity. When the weight of the partially pure sampleis approximately 0.5 g or less, countercurrent chromatography is carriedout on all Ito multi-layered horizontal Coil Planet Centrifuge (P.C.Inc., Potomac, Md.) using either the system consisting of n-hexane,ethyl acetate, 0.02 M aqueous potassium phosphate, with the pH chosenbetween 6.5-8.0, (3:7:5, v/v/v) employed above, or similar systems inwhich the ratio of hexane to EtOAc and/or the pH are varied. Thechromatography is developed either isocratically, or with a gradientstarting, for example, with the upper phase of a solvent systemconsisting of n-hexane, ethyl acetate, 0.02 M aqueous potassiumphosphate, with the pH chosen between 6.5-8.0, (7:3:5, v/v/v) andfinishing with the upper phase of a solvent system consisting ofn-hexane, ethyl acetate, 0.02 M aqueous potassium phosphate, at the samepH, (1:1:1, v/v/v). In all cases, flow rates of approximately 120 mL/hrare employed. As before, fractions are analyzed by NMR bioactivity, andpooled according to purity. Once sufficient purity is achieved, ¹H and¹³C NMR spectra are measured with a General Electric GN500 spectrometerand structural assignments are made with the aid of with the aid ofcorrelational spectroscopy (COSY), heteronuclear multiple quantumcorrelation (HMQC), heteronuclear multiple bond correlation (HMBC), anddistortionless enhancement by polarization transfer (DEPT) experiments.

[0147] The foregoing can be better understood by reference to thefollowing examples, which are provided as non-limiting illustrations ofthe practice of the instant invention.

EXAMPLE 1 Cloninig of the LigAT2 Domain from Streptomyces hygroscopicusATCC 29253

[0148] A genomic library of Streptomyces hygroscopicus ATCC 29253 DNAwas constructed in the bifunctional cosmid pNJ1 (Tuan, et al., Gene 90:21-29 (1990)) using standard methods of recombinant DNA technology.Briefly, cosmid vector was prepared by digesting approximately 5 μg ofpNJ1 with EcoRI, dephosphorylating with calf intestinal alkalinephospatase (CIAP) and then digesting with Bg;II to generate one arm andalso digesting 5 μg of pNJ1 with HindIII, dephosporylating with CIAP andthen digesting with BglII to generate the other. Insert DNA was preparedby partially digesting approximately 25 μg of high molecular wight S.hygroscopicus chromosomal DNA with SauIIIA according to the procedureoutline in Maniatis, et al. supra. SauIIIA fragments of approximately 35kb were recovered from a 0.5% low melting point agarose gel by meltingthe appropriate gel slice to 65° C., adding 3 volumes of TE buffer,gently extracting 2X with phenol and once with chloroform and ethanolprecipitating the aqueous phase. For the ligation, approximately 3 μg ofthis chromosomal DNA was mixed with approximately 0.5 μg of each cosmidarm and EtOH precipitated. The precipitate was resuspended in 7 μL, ofwater to which was added 2 μL of 5X ligation buffer and 1 μL of T4 DNAligase. The mixture was incubated overnight at 16° C. GigapackII XL(Stratagene®) was used for packaging 2 μL of the ligation mix accordingto the manufacture's instructions. The host bacterium was E. coli ER1772 from New England Biolabs (Beverly, Mass.). Twenty-six colonies wereexamined by restriction analysis and all were found to contain insertDNA. Individual colonies were picked into thirty-four 96-well plates togive a 99.99% probability that the library represented all S.hygroscopicus sequences. Further restriction analysis demonstrated theaverage insert size to be about 30 kb.

[0149] The library was screened with a 1.45 kb SstI-MscI DNA fragmentencompassing the ketosynthase (KS) domain from module 5 of theerythromycin PKS gene eryAIIl (Donadio and Katz, 1992, Gene, 111:51-60). The DNA fragment was labeled with ³²P using the Megaprime DNAlabeling system (Amersham Life Science, Arlington Heights, Ill.).Colonies (3600) were transferred from 96-well plates to Hybond-N nylonmembranes (Amersham Life Science, Arlington Heights, Ill.) and probedaccording to procedures outlined in Maniatis, et al. supra.Hybridization was performed at 65° C. and a stringency wash carried outwith 0.1×SSC at 65° C. About 60 cosmid clones were chosen which gave thestrongest signals with this PKS probe.

[0150] We also decided to screen Southern digests of these clones with asecond probe in order to identify potential genetically linked peptidesynthetases in this strain. The probe was designed from conserved motifsof nonribosomal peptide synthetases (Borchert et al., 1992, FEMSMicrobiology Letters, 92: 175-180) and consisted of a mixture of twodegenerative 35-mers, SEQ ID NO:3 and SEQ ID NO:4. The mixed probe waslabeled using DNA 5′ end Labeling System (Promega Corp., Madison, Wis.).The 60 cosmid clones were digested with SmaI and run on 0.9% agarosegels. Southern analysis was performed according to Maniatis, et al.supra, except that hybridization was overnight at 55° C. and thestringency wash was with 0.5×SSC at 55° C. Two cosmids, 54 and 58, wereidentified using this second probe. Thirteen additional cosmids weresubsequently isolated by re-probing the cosmid library with a 1 kbfragment from the left of the insert of cosmid 58. Two of these thirteencosmids, sequencing. Restriction and sequence analysis of a 32.8 kbcontinuous segment of DNA from A16 revealed a type I PKS cluster withfour PKS modules. A genetic map of the cluster is shown in FIG. 6. Sincean unusual CoA ligase-like domain was found in ORFI (PKS I), the clusterwas named “Lig-PKS”.

[0151] The nucleotide sequence of the LigAT2 domain from Lig-PKS (topstrand) and its corresponding amino acid sequence (bottom strand) areshown in FIG. 7 (SEQ ID NO:1 and SEQ ID NO:31 respectively). When SEQ IDNO:31 was compared with the 14 AT domains in the rapamycin PKS (GrowtreeProgram, GCG, Madison Wiss.), it was found to cluster withmalonate-specifying rapamycin domains (see Growtree analysis of FIG. 3).Therefore, it was predicted that the LigAT2 specifies malonate as itscognate extender unit during synthesis of the polyketide encoded byLig-PKS.

EXAMPLE 2 Construction of plasmid pUC18/LigAT2

[0152] Two PCR oligonucleotides (SEQ ID NO:5 and SEQ ID NO:6) weredesigned to subclone the 985 bp DNA segment encoding the LigAT12 domainfrom the Lig-PKS cluster and to introduce two unique restriction sites,AvrII, and NsiI, for cassette cloning. The unique restriction sitesAvrII and NsiI required for cassette cloning of the AT-encoding DNA werechosen based on multiple sequence alignment using the programs PILEUPand PRETTY (GCG, Madison Wiss.) which compared the amino acid sequencesof LigAT2, venAT, rapAT2, rapAT5, rapAT8, rapAT9, rapAT11, rapAT12,rapAT14, eryAT1, eryAT2, eryAT3, eryAT4, eryAT5, eryAT6, and amonofunctional AT from Steptomyces glaucescens (R. G. Summers et al.,Biochemistry 34: 9389-9402 (1995)). The selection and positioning of therestriction enzymne sites were based on the following considerations:(i) extent of amino acid sequence conservation among the various ATs,with the sites being positioned outside, but near the regions ofgreatest conservation, (ii) absencce of the sites from the heterologousAT-encoding DNA and the eryAT flanking DNA and (iii) impact of the aminoacid sequence changes resulting from translation of these sites on theheterologous AT amino acid sequence. This necessitated nucleotidechanges, shown in bold in FIG. 8, at the beginning and near the end ofthe LigAT2-encoding DNA sequence. (In FIG. 8, the underlined nucleotidesare the wild-type sequence.) In addition, two other restriction sites,EcoRI and BamHI, were also introduced at the 5′ ends of the N-terminaland C-terminal oligonucleotides, respectively, for convenient subcloningof the PCR-generated product. The approximately 1 kb LigAT2 domain wasamplified from Cosmid 58 as follows: The 100 μL PCR reaction mixturecontained 10 μL of 10× PCR buffer (Bethesda Research Laboratories), 2 μLof 10 mM dNTP mixture, 2-4 μL of 50 mM MgCl_(2, 100) pM of each oligo,10-50 ng of template DNA and water to 100 μL. Cycling conditions were asfollows: One cycle at 96° C./6 min, 80° C./1 min (add 5 U Taq DNAPolymerase during this 1 min) and 72° C./2 min; 30 cycles at 95° C./1min, 65° C./1 min and 72° C./2 min with a 5 min extension at 72° C. forthe last cycle. The entire reaction was then run on a 1% agarose gel andthe desired fragment was isolated with Prep-A-Gene (BioRad, Hercules,Calif.). The PCR product was digested with EcoRI and BamHI andsubclonedl into the EcoRI and BamHI sites of pUC18. The ligation mixturewas transformed into E. coli DH5α (GIBCO BRL) according to themanufacturer's instructions and transformants were selected on LB platescontaining 150 μg/ml, ampicillin and 50 μL of a 2% solution of X-gal forblue/white selection. Clones were confirmed by restriction analysis andthe fidelity of the insert was confirmed by DNA sequencing. The finalplasmid construct was named pUC18/LigAT2.

EXAMPLE 3 Construction of plasmid pEryAT1/LigAT2

[0153] pEryAT1/LigAT2 was constructed using standard methods ofrecombinant DNA technology according to the schematic outlines of FIGS.9 and 10. To construct it gene-replacement vector specific for theeryAT1 domain, the two DNA regions immediately adjacent toeryAT1-encoding DNA were cloned and positioned adjacent to theLigAT2-encoding DNA as described in Example 2. The 5′ and 3′ boundariesof eryAT1 were designated as 3825 and 4866, and corresponded to thedeposited eryAl sequence (GenBank accession number M63676). To subclonethe DNA fragment upstream of the eryAT1 domain encoding region from theSac. erythraea chromosome, two PCR oligonucleotides (SEQ ID NO:7 and SEQID NO:8) were designed so that an EcoRI site was added at the 5′ end ofthe region and AvrII-BamHI restriction sites were introduced at the 3′end. The 5′-flanking region (about 1 kb) was PCR generated as describedin EXAMPLE 2 using plasmid pAIEN22 DNA as template. (This plasmid is apUC19 derivative containing 22 kb of Sac. erythraea DNA from an EcoRIsite upstream of eryAI to an NheI site in eryAII cloned into EcoRI andXbaI cut pUC19). The PCR product was subcloned into EcoRI and BamHIsites of pUC19 and the ligated DNA transformed into E. coli DH5α (GIBCOBRL) according to the manufacturer's instructions. Clones were selectedon LB plates containing 150 μg/ml, ampicillin and 50 μL of a 2% solutionof X-gal for blue/white selection. Clones were confirmed by restrictionanalysis and the fidelity of the insert was confirmed by DNA sequencing.The resulting construct was named pUC19/AT1/5′-flank.

[0154] For subcloning the 3′-flanking-region of the eryAT1 from Sac.erythraea chromosome, two PCR oligoniucleotides (SEQ ID NO:9 and SEQ IDNO:10) were designed so that BamHI-Nsil restriction sites wereintroduced into the 5′ end of the region and a HindIII restriction sitewas added to the 3′ end. The 3′-flanking region (about 1 kb) was alsogenerated by PCR using pAIEN22 as template as described above. The PCRfragment was subcloned into the BamHI and HindIII sites of pUC19 and theligated DNA transformed into E. coli DH5α as above. Clones were selectedon LB plates containing 150 μg/ml ampicillin and 50 μL of a 2% solutionof X-gal for blue/white selection. Clones were confirmed by restrictionanalysis and the fidelity ot the insert was confirmed by DNA sequencing.This intermediate construct was named pUC19/AT1/3′-flank. The twoflanking regions were joined by first isolating the 1 kb BamHI-HindIIIfragment (3′-flank) from pUC19/AT1/3′-flank and then ligating thisfragment to pUC19/AT1/5′-flank cut with BamHI and HindIII. Ligated DNAwas transformed into E. coli DH5α and clones isolated as described. Theresulting plasmid was named pUC19/AT1-flank. The 2.1 kb EcoRI andHindIII fragment from pUC19/AT1-flank was then isolated and ligated topCS5 cut with the same enzymes to generate pCS5/AT1-flank. The finalstep in the construction of pEryAT1/LigAT2 was to ligate the 1 kb LigAT2fragment having AvrII and NsiI ends to pCS5/AT1-flank cut with the sameenzymes to give the gene replacement/integration plasmid pEryAT1/LigAT2.All ligation mixtures were transformed into the intermediate host E.coli DH5α and clones selected as previously described.

EXAMPLE 4 Construction of Sac. erythraea ER720 EryAT1/LigAT2

[0155] An example of a 12-desmethyl-12-deoxyerythromycin A producingmicroorganism was prepared by replacing the DNA fragment encoding themethylmalonyl acyltransferase domain in module 1 of the erythromycin PKS(EryAT1) of Sac. erythraea ER720 with a newly discovered DNA fragmentencoding a malonyl acyltransferase domain (LigAT2) from S. hygroscopicusATCC 29253. This was accomplished with the recombinant plasmid,pEryAT1/LigAT2, prepared as described in Example 3. Transformation ofSac. erythraea ER720 and resolution of the integration event werecarried out according to the following method. Sac. erythraea ER720cells were grown in 50 mL of SGGP medium (per 1 liter aqueous solution:4 g peptone, 4 g yeast extract, 4 g casamino acids, 2 g glycine, 5 gMgSO₄.7H₂ 0, 10 g glucose, 20 mL of 500 mM KH₂PO₄) for 3 days at 32° C.and then washed in 10 mL of 10.3% sucrose. The cells were resuspended in10 ml of P_(M) buffer containing 1 mg/mL lysozyme and incubated at 30°C. for 15-30 minutes until most of the mycelial segments were convertedinto spherical protoplasts. (P_(M) buffer per 1 liter aqueous solution:200 g sucrose, 0.25 g K₂SO₄ in 890 ml H₂O, with the addition aftersterilization of 100 mL 0.25 M TES, pH 7.2, 2 mL trace elements solution(Hopwood, et al., 1985, Genentic Manipulation of Streptomyces ALaboratory Manual, The John Innes Foundation), 0.08 mL 2.5 MCaCl_(2, 10) mL 0.5% KH₂PO₄, 2 mL 2.5 M MgCl₂.) The protoplasts werewashed once with PM and then resuspended in 3 mL of the same buffercontaining 10% DMSO for storage in 200 μL aliquots at ×80° C.

[0156] Transformation was accomplished by quickly thawing an aliquot ofprotoplasts, centrifuging for 15 seconds in a microfuge, decanting thesupernatant, and resuspending the protoplasts in the P_(M) remaining inthe tube. Ten μL of DNA solution was added (3 μL of pEryAT1/LigAT2 DNAfrom Example 3 at about 1 μg/μL in 7 μL of P_(M) buffer) and mixed withthe protoplasts by gently tapping the tube. Two tenths of a mL of 25%PEG 8000 in T buffer (Hopwood, et al., 1985, Genetic Manipulation ofStreptomyces A Laboratory Manual, The John Innes Institute) was thenadded, mixed by pipetting the solution 3 times and the suspensioninmmediately spread on a dried R3M plate. The plate was incubated at 30°C. for 20 hours and overlaid with 2 mL of water containing 100 μg/mLthiostrepton, dried briefly and incubated 4 more days at 30° C.

[0157] To select stable transormants (integrants) colonies arising onthe transformation plate were re-streaked onto R3M plates containingthiostreption (20 μg/mL). Two colonies were confirmed to be thiostreptonresistant and one of these was inoculated into SGGP containingthiostrepton (10 μg/mL) to isolate chromosomal DNA for Southernanalysis. Integration of the plasmid DNA into the ER720 chromosome wasfurther confirmed by Southern hybridization (data not shown).Hybridization was at 65° C. and the stringency wash was with 0.1×SSC at65° C.

[0158] The confirmed integrant was grown in SGGP without antibiotic forfour days and then plated onto non-selective R3M plates for sporulation.Spores were plated on R3M plates to obtain individual colonies, whichwere then screened for sensitivity to thiostrepton, indicating loss ofthe plasmid sequence from the chromosome. Five thiostrepton sensitivecolonies were selected and 3 of these were confirmed by Southernhybridization to have the EryAT1 replaced by LigAT2 (FIG. 11).Hybridization was at 65° C. and the stringency was was with 0.1×SSC at65° C. The strain was named Sac. erythraea ER720 EryAT1/LigAT2.

EXAMPLE 5 Analysis of compounds produced by Sac. erythraea ER720EryAT1/LigAT2

[0159] Compounds produced by the recombinant Sac. erythraea strain,ER720 EryAT1/LigAT2, whose construction is described in Example 4, werecharacterized by TLC, bioautography, mass spectrometry and NMR analysis.

[0160] For TLC analysis cells were grown in either SGGP or SCM medium(20 g Soytone, 15 g Soluble Starch, 10.5 g MOPS, 1.5 g Yeast Extract and0.1 g CaCl₂ per liter of distilled H₂O) for 4-5 days at 30° C. 1.5 mL ofculture was centrifuged for 1 minute in a microfuge to remove cells. OnemL of the resulting supernatant was removed to another microfuge tubeand the pH adjusted to 9.0 by the addition of 6 μL of NH_(4OH. Then) 0.5mL of ethyl acetate was added, the tube was vortexed for 10 sec and thencentrifuged for approximately 5 min to achieve phase separation. Theorganic phase was removed to another tube, and the aqueous phase wasre-extracted with 0.5 mL of ethyl acetate. The second organic phase wascombined with the first and dried in a Speed Vac. The residue was takenup in 10 μL of ethyl acetate and 5 μL was spotted onto a Merck 60F-254silica gel TLC plate. The plate was run in isopropyl ethermethanol:NH₄OH(75:35:2). Erythromycin derivatives were visualized by spraying theplates with anisaldehyde:sulfuric acid:ethanol (1:1:9). Using thisreagent, a novel compound predicted to be12-desmethyl-12-deoxyerythromycin A, appeared as a blue spot runningslightly faster than erythromycin A (FIG. 12).

[0161] To detect biological activity, a TLC-bioautography assay wasperformed. In this assay, one microliter of the extracted sample fromabove was spotted onto a TLC plate which was run as described above. Theplate was then air-dried and placed in a sterile bio-assay dish(245×245×25 mm). The plate was then covered with 100 mL of antibioticmedium II (DIFCO-BACTO) containing Stephylococcus aureus as an indicatorstrain and incubated overnight at 37° C. As with the positive controls,a clear zone of inhibition developed around the sample spot indicatingthat the novel compound had bioactivity.

[0162] To determine whether the novel spot seen on TLC had the molecularmass corresponding to the predicted 12-desmethyl-12-deoxyerythromycin A,an ethyl acetate extract was further analyzed by mass spectrometry. Themass spectrometry samples were isolatetd by TLC basically as describedabove except that plates were not sprayed with the anisaldehyde reagent.The region of the novel spot was instead scraped from the TLC plate andthe silica resin re-extracted with ethyl acetate-methanol (1:1) and thentwice with ethyl acetate. The combined solvent phases were then dried ina Speed Vac. Mass spectrometric analysis revealed the novel compound toleave a mass of 704, which corresponds to the molecular ion plus aproton (M+H⁺) of 12-desmethyl-12-deoxyerythromycin A.

[0163] To acquire milligram quantities of highly purified material forperformance of NMR analysis, the culture was grown in a 42-liter LHFermentation Series 2000 fermentor. SCM medium was used for growth ofinoculum and for the fermentation. Seed for the fermentation was grownin two steps. In the first step, frozen vegetative inoculum was used toseed 100 mL of SCM medium in a 500 mL Erlenmeyer flask. For the secondstep, 2-liter Erlenmeyer flasks containing 600 mL of SCM medium wereseeded at 5% from the first passage growth. Each step was incubated for3 days at 32° C. on a rotary shaker operated at 225 rpm.

[0164] Thirty liters of SCM medium were prepared in the 42-literfermentor and sterilized at 121° C. and 15 psi for 1 hour. Antifoam(XFO-371, Ivanhoe Chemical Co., Mundelein, Ill.) was addled initially at0.01% and then was available on demand. THe fermentor was inoculatedwith 1.5 liters of the second passage seed growth. The temperature wascontrolled at 32° C. The agitation rate was 260 rpm and the air flow was1.3 vol/vol/min. The head pressure was maintained at 6 psi. Duringfermentation pH was controlled at 7.3 with 5 M propionic acid. Thefermentation was terminated at 111 hours, and the fermentation beer wasadjusted to pH 8. This was followed by two extractions with equalvolumes of CH₂Cl₂. The pooled CH₂Cl₂ extract was then concentrated toapproximately 400 mL and extracted twice with equal volumes of 0.05 Maqueous potassium phosphate pH 5.5. The aqueous phase was pooled andadjusted to pH 8, and then extracted twice with equal volumes of ethylacetate. The ethyl acetate extracts were pooled and concentrated toyield 5 ml oil. The extraction sequence described above was thenrepeated to yield 600 mg of oil after concentration. Next, the samplewas split and each half was digested in 2.5 ml each of the upper andlower phases of a solvent system consisting of n-hexane, ethyl acetate,0.02 M aqueous potassium phosphate, pH8, (1:1:1, v/v/v). These were thenchromatographed on the Coil Planet Centrifuge using the upper phase asthe mobile phase. Fractions were analyzed by bioassay againstStaphylococcus aureus and ¹H NMR. Two macrolide containing peaks ofbioactivity were observed in both samples, and the later eluting peaksfrom each sample, which contained most of the bioactivity, were pooledand concentrated. The concentrated material was then digested in 2.5 mLeach of the upper and lower phases of a solvent system consisting ofn-hexane, ethyl acetate, 0.02 M aqueous potassium phosphate, pH 6.5,(6:4:5, v/v/v), and was chromatographed on the Coil Planet Centrifugeusing the upper phase as the mobile phase. Fractions were analyzed bybioassay and ¹H NMR. Two macrolide containing peaks of bioactivity wereobserved and the later eluting species was readily characterized by its¹H and ¹³C NMR spectra as 12-desmethyl-12-deoxyerythromyciln A.Parameters from the ¹H NMR spectra are listed in Table 2. Theassignments were made with the aid of correlational spectroscopy (COSY),heteronuclear multiple quantum correlation (HMQC), heteronuclearmultiple bond correlation (HMBC), and distortionless enhancement bypolarization transfer (DEPT) experiments. Mass spectral data of thissample was also consistent with the structural assignment. Electrosprayionization (ESI) of this sample revealed an M+H⁺ion at M/Z 704, which isin full accord with erythromycin A lacking both a methyl group and ahydroxyl group. TABLE 2 ¹H NMR chemical shift (∂) assignments for12-desmethyl-12-deoxyerythromycin A in CDCl₃  2-H 2.74 1′-H 4.47  3-H4.15 2′-H 3.25  4-H 2.01 3′-H 2.49  5-H 3.58 4′-H_(a) 1.67  7-H_(a) 1.914′-H_(b) 1.23  7-H_(b) 1.66 5′-H 3.54  8-H 2.86 6′-H₃ 1.23 10-H 2.70N(CH₃)₂ 2.30 11-H 4.05 1″-H 4.85 12-H_(a) 1.71 2″-H_(a) 2.40 12-H_(b)1.46 2″-H_(b) 1.59 13-H 5.06 4″-H 3.03 14-H₂ 1.59 5″-H 4.04 15-H₃ 0.896″-H₃ 1.30  2-CH₃ 1.19 3″-CH₃ 1.25  4-CH₃ 1.13 OCH₃ 3.33  6-CH₃ 1.38 8-CH₃ 1.19 10-CH₃ 1.11

EXAMPLE 6 Construction of plasmid pEryAT2/LigAT2

[0165] pEryAT2/LigAT2 was constructed using standard methods ofrecombinant DNA technology. To make a gene-replacement vector specificfor the eryAT2 domain, two DNA regions flanking eryAT2 were cloned andpositioned adjacent to the DNA encoding the domain to be inserted inorder to effect homologous recombination. Boundaries of the AT2 domainwere chosen as described in Example 2. The 5′ and 3′ boundaries oferyAT2 are designated as 8255 and 9282, respectively, and correspond todeposited eryAl sequence (GenBank accession number M63676). To subclonethe DNA fragment upstream of the eryAT2 DNA, two PCR oligonucleotides(SEQ ID NO:11 and SEQ ID NO:12) were designed so that a HindIII site wasadded at the 5′ end of the region and AvrII-PstI restriction sites wereintroduced at the 3′ end. For subcloninig the 3′-flanking region oferyAT2, two PCR oligonucleotides (SEQ ID NO:13 and SEQ ID NO:14) weredesigned so that PstI-NsiI restriction sites were introduced at the 5′end of the region and an EcoRI site at the 3′ end. Both the 5′-flankingand 3′-flanking regions (about 1 kb each) were PCR generated asdescribed in Example 3. In the case of the 5′-flanking region, the PCRproduct was subsequently subcloned into HindIII and PstI sites of pUC18whereas the PCR product of the 3′-flanking region was subcloned into thePstI and EcoRI sites of pUC18. Ligations, transformations andconfirmations of selected clones were performed as in Example 3. Theresulting construct containing the AT2 5′-flanking region was designatedpUC18/AT2/5′-flank and the construction containing the AT2 3′-flankingregion was designated pUC18/AT2/3′-flank. The two flanking regions werethen joined by first isolating the 1 kb PstI and EcoRI fragment(3′-flank) from pUC18/AT2/3′-flank, and ligating this fragment topUC18/AT2/5′-flank cut with PstI and EcoRI. The ligation was transformedinto E. coli DH5α and clones isolated as described. The resultingplasmid was named pUC18/AT2-flank (FIG. 13). The 2.2 kb EcoRI andHindIll fragment from pUC18/AT2-flank was then isolated and ligated topCS5 cut with the same enzymes to generate pCS5/AT2-flank. The finalstep in the construction of pEryAT2/LigAT2 was to ligate the LigAT2encoding DNA fragment from pUC18/LigAT2 having AvrII and NsiI ends(described in Example 2) to pCS5/AT2-flank cut with the same enzymes togive the gene replacement, integration plasmid pEryAT2/LigAT2 (FIG. 14).All ligations were transformed into the intermediate host E. coli DH5αand clones selected as previously described.

EXAMPLE 7 Construction of Sac. erythraea ER720 EryAT2/LigAT2

[0166] An example of a 10-desmethylerythromycin A and10-desmethyl-12-deoxyerythromycin A producing microorganism was preparedby replacing the methylmalonyl acyltransferase domain of module 2 of theerythromycin PKS (EryAT2) of Sac. erythraea ER720 with a newlydiscovered malonyl acyltransferase domain (LigAT2) from S. hygroscopicusATCC 29253. This was accomplished with the recombinant plasmid,pEryAT2/LigAT2, prepared as described in Example 6. Transformation ofER720 and resolution of the integration event were carried out accordingto the procedures described in Example 4 using 10 μL of a DNA solutionconsisting of 3 μL of pEryAT2/LigAT2 DNA from Example 6 at about 1 μg/μLin 7 μL of P_(M) buffer. Three colonies were confirmed to bethiostrepton resistant and were inoculated into SGGP containingthiostrepton (10 μg/mL) to isolate chromosomal DNA for Southernanalysis. Integration of the plasmid DNA into ER720 chromosome wasfurther confirmed by Southern hybridization (data not shown).Hybridization was at 65° C. and the stringency wash was with 0.1×SSC at65° C.

[0167] The confirmed integrant was grown in SGGP without antibiotic forfour days and then plated onto non-selective R3M plates for sporulation.Spores were plated on R3M plates to obtain individual colonies, whichwere then screened for sensitivity to thiostrepton, indicating loss ofthe plasmid sequence from the chromosome. Two thiostrepton sensitivecolonies were selected and one of these was confirmed by Southernhybridization to have the EryAT2 replaced by LigAT2 (FIG. 15).Hybridization was at 65° C. and the stringency wash was with 0.1×SSC at65° C. The strain was named Sac. erythraea ER720 EryAT2/LigAT2.

EXAMPLE 8 Analysis of compounds produced by Sac. erythraea ER720ErvAT2/LigAT2

[0168] Compounds produced by the recombinant Sac. erythraea strain,ER720 EryAT2/LigAT2, whose construction is described in Example 7, werecharacterized by TLC, bioautography and mass spectrometry.

[0169] For small scale analysis, the cells were grown in either SGGP orSCM medium for 4-5 days at 30° C. 15 mL of culture was centrifuged for10 minute in a Sorval GLC-4 General Laboratory Centrifuge at setting 10to remove cells. Ten mL of the resulting supernatant was pH adjusted to9.0 by the addition of 60 μL, NH₄OH. Then 5 ml of ethyl acetate wasadded, the tube was shaken vigorously for 3 minutes and then centrifugedfor approximately 5 min to achieve phase separation. The organic phasewas removed to another tube, and the aqueous phase was re-extracted with5 mL of ethyl acetate. The second organic phase was combined with thefirst and dried in a Speed Vac. The residue was taken up in 20 μL ofethyl acetate and 10 μL was spotted onto a Merck 60F-254 silica gel TLCplate. The plate was run in isopropyl ether:methanol:N₄OH (75:35:2).Erythromycin derivatives were visualized by spraying the plates withanisaldehyde:sulfuric acid:ethanol (1:1:9). Using this reagent, twonovel compounds predicted to be 10-desmethylerythromycin A and10-desmethyl-12-deoxyerythromycin A, appeared as blue spots with thelower spot running slightly slower than erythromycin A and( upper spotrunning slightly faster than erythromycin A (FIG 16).

[0170] To detect biological activity, a TLC-bioautography assay wasperformed. In this assay, 0.2 to 1 microliter of the extracted samplefrom above was spotted onto a TLC plate which was run as described. Theplate was then air-dried and placed in a sterile bio-assay dish(245×245×25 mm). The plate was then covered with 100 mL of antibioticmedium II (DIFCO-BACTO) containing Staphylococcus aureus as an indicatorstrain. The inihibition zones were developed by overnight incubation ofthe plate at 37° C. As shown in FIG. 17 (TLC-bioautography), the twonovel spots (compounds) each have bioactivity against Staphylococcusaureus.

[0171] To determine whether the novel spots seen on TLC had themolecular masses corresponding to the predicted 10-desmethylerythromycinA and 10-desmethyl-12-deoxyerythromycin A, all ethyl acetate extract wasfurther analyzed by mass spectrometry. The mass spectrometry sampleswere isolated by TLC similarly to the method described above except thatplates were not sprayed with the anisaldehyde reagent. Instead, tworegions which contain the novel spots were scraped frown the TLC plateand the silica resin re-extracted with ethyl acetate-methanol (1:1) andthen twice with ethyl acetate. The combined solvent phases were thendried in a Speed Vac. In addition to the samples described above, acrude ethyl acetate extract was also analyzed by LC-MS, in which thesample components were first separated by liquid chromatography and thenanalyzed by mass spectrometry. Mass spectrometric analysis revealed thetwo novel compounds to have masses of 720 and 704, which correspond tothe molecular ion plus a proton (M+H⁺) of 10-desmethylerythromycin A and10-desmethyl-12-deoxyerythromycin A, respectively.

EXAMPLE 9 Cloning of the venAT Domain from Streptomyces venezuelae

[0172] A genomic library of Streptomyces venezuelae ATCC 15439 DNA wasconstructed in the bifunctional cosmid pNJ1 (Tuian, et al., Gene 90:21-29 (1990)) using standard methods of recombinant DNA technology. Acosmid from this library, pVen17, was characterized by Southern analysisand SstI fragments of approximately 3.5, 3.8, and 4.0 kb were found tohybridize to a 1.37 kb SmaI fragment that encompasses the ketosynthase(KS) domain from module 2 of the erythromycin PKS gene eryAl (Donadio etal., Science 252: 675-679 (1991)). The 4.0 kb SstI fragment was thensubcloned into pUC19 to give pVen4.0. The nucleotide sequence of pVen4.0insert DNA was determined from single straid DNA templates prepared fromM13mp and M13mp19 (Yanisch-Perron, et al., Gene, 33: 103 (1985))subclones using Sequenase version 2.0 with 7-deaza-dGTP (United StatesBiochemical, Cleveland, Ohio) and 5′-|α-³²P| or 5′-α³³P|-dCTP (NENResearch Products, Boston, Mass.). Because pVen4.0 did not contain theentire AT domain, the nucleotide sequence was extended using pVen17 DNAas the template. The nucleotide sequence of the venAT domain (SEQ IDNO:2) and its corresponding amino acid sequence (SEQ ID NO:32) is shownin FIG. 18 (top and bottom strands respectively).

EXAMPLE 10 Construction of plasmid pEryAT1/venAT

[0173] pEryAT1/venAT was constructed using standard methods ofrecombinant DNA technology according to the schematic outlines of FIGS.23 , and 24. Two PCR oligonucleotides (SEQ ID NO:15 and SEQ ID NO:16)were designed to subclone the 1.03 kb DNA fragment that encodes thevenAT domain (FIG. 19) from the S. venezuelae PKS cluster and tointroduce two unique restriction sites, AvrIIl and NsiI, for cassettecloning (described in Example 2). This necessitated nucleotide changes(shown in bold in FIG. 19) at the beginning and near the end of thevenAT sequence (underlined nucleotides are the wild-type sequence). Inaddition, two other restriction sites, EcoRI and BamHI, were alsointroduced at the 5′ ends of the N-terminal and C-terminialoligonucleotides, respectively, for convenient subcloning of thePCR-generated product. The approximately 1 kb venAT-encodiing DNA wasPCR amplified from cosmid pVen17 template DNA (Example 2) usingVent_(R)® DNA Polymerase (New England Biolabs). A typical PCR reactioncontained 10 μL ThermoPol Buffer, 10 μL formamide, 10 μL of 20%glycerol, 55 μL water, 100 pmole of each primer, digested pUC18 andtransformed into E. coli DH5α (GIBCO BRL) according to themanufacturer's instructions. Clones were selected on LB platescontaining 150 μg/mL ampicillin and 50 μL of a 2% solution of X-gal forblue/white selection. Clones were confirmed by restriction analysis andthe fidelity of the insert was confirmed by DNA sequencing. The finalconstruct was named pUC18/venAT.

[0174] The final step in the construction of pEryAT1/venAT was to ligatethe 1 kb venAT fragment having AvrII and NsiI ends to pCS5/AT1-flank(Example 3) cut with the same enzymes to give the genereplacement/integration plasmid pEryAT1/venAT (FIG. 20). All ligationswere transformed into the intermediate host E. coli DH5α and clonesselected as previously described.

EXAMPLE 11 Construction of Sac. erythraea ER720 EryAT1/venAT

[0175] A 12-desmethyl-2-deoxyerythromycin A producing microorganism wasprepared by replacing the methylmalonyl acyltransferase domain of module1 of the erythromycin PKS (EryAT1) of Sac. erythraea ER720 with a newlydiscovered malonyl acyltransferase domain (venAT) from S. venezuelaeATCC 15439. This was accomplished with the recombinant plasmid,pEryAT1/venAT, prepared as in Example 10. Transformation of ER720 andresolution of the integration event were carried out as described inExample 4 using 10μL of DNA solution consisting of 3 μL of pEryAT1/venATDNA at about 1 μg/mL in 7 μL of P_(M) buffer. One thiostrepton resistantcolony was isolated and was inoculated into SGGP containing thiostrepton(10 μg/mL) to isolate chromosomal DNA for Southern analysis. Integrationof the plasmid DNA into the ER720chromosome was further confirmed bySouthern hybridization (data not shown). Hybridization was at 65° C. andthe stringency wash was with 0.1×SSC at 65° C.

[0176] The confirmed integrant was grown in SGGP without antibiotic forfour days and then diluted 1000 fold into fresh medium and grown for 4more days. Cells were then plated onto non-selective R3M plates forsporulation. Spores were plated on R3M plates to obtain individualcolonies, which were then screened for sensitivity to thiostrepton,indicating loss of the plasmid sequence from the chromosome. Fourthiostrepton sensitive colonies were selected and 2 of these wereconfirmed by Southern hybridization, using conditions described above,to have the EryAT1 replaced by venAT (FIG. 21). The strain was namedSac. erythraea ER720 EryAT1/venAT.

EXAMPLE 12 Analysis of compounds produced by Sac. erythraea ER720EryAT1/venAT

[0177] Compounds produced by the recombinant Sac. erythraea strain,ER720 EryAT1/venAT, whose construction is described in Example 11, werecharacterized by TLC, bioautography, and mass spectrometry.

[0178] For TLC analysis cells were grown in either SGGP or SCM medium(20 g Soytone, 15 g Soluble Starch, 10.5 g MOPS, 1.5 g Yeast Extract and0.1 g CaCl2 per liter of distilled H₂) for 4-5 days at 30° C. Theculture (1.5 InL) was centrifuged for 1 minute in a microfuge to removecells. One mL of the resulting supernatant was removed to anothermicrofuge tube and the pH adjusted to 9.0 by the addition of 6 μL ofNH4OH. Then 0.5 mL of ethyl acetate was added, the tube was vortexed for10sec and then centrifuged for approximately 5 min to achieve phaseseparation. The organic phase was removed to another tube, and theaqueous phase was re-extracted with 0.5 mL of ethyl acetate. The secondorganic phase was combined with the first and dried in a Speed Vac. Theresidue was taken up in 10 μL of ethyl acetate and the entire sample wasspotted onto a Merck 6OF-254 silica gel TLC plate. The plate was run inisopropyl ether:methanol:NH4OH (75:35:2). Erythromycin derivatives werevisualized by spraying the plates with anisaldehyde:sulfuricacid:ethanol (1:1:9). Using this reagent, a novel compound predicted tobe 12-desmethyl-12-deoxyerythromycin A, appeared as a blue spot runningslightly faster than erythiomycin A (FIG. 22).

[0179] To detect biological activity, a TLC-bioautography assay wasperformed. In this assay, one μL of an extract prepared as above wasspotted onto a TLC plate which was run as described above. The plate wasthen air-dried, placed face down on top of 100 mL of antibiotic mediumII (DIFCO-BACTO) containing Staphylococcus aureus as an indicator strainin a sterile bio-assay dish (245×245×25 mm) and incubated overnight atat 37° C. As with the positive controls, a clear zone of inhibitiondeveloped around the sample spot indicating that the novel compounid wasbioactive.

[0180] To determine whether the novel spot seen on TLc had the molecularmass corresponding to the predicted 12-desmethyl-12-deoxyerythromycin A,an ethyl acetate extract was further analyzed by mass spectrometry. Themass spec samples were isolated by TLC basically as described aboveexcept that plates were not sprayed with anisaldelhyde. The region ofthe novel spot was instead scraped from the TLC plate and the silicaresin re-extracted with ethyl acetate-methanol (2:1) and then twice withethyl acetate. The combined solvent phases were then dried in a SpeedVac. Mass spectrometric analysis revealed the novel compound to have amass of 704, which corresponds to the molecular ion plus a proton (M+H⁺)of 12-desmethyl-12-deoxyerythromycin A.

EXAMPLE 13 Construction of plasmid pUC19/rapAT14

[0181] Two PCR oligonucleotides (SEQ ID NO:17 and SEQ ID NO:18) weredesigned to subclone the 1023 bp rapAT14-encodinig DNA fragment from therapamycin biosynthetic gene cluster (GenBank Accession #: X86780) and tointroduce two unique restriction sites, AvrII and NsiI, for cassettecloning (described in Example 2). This necessitated nucleotide changes(shown in bold in FIG. 23) at the beginning and near the end of therapAT14 sequence. (In FIG. 23, the underlined nucleotides are thewild-type sequence.) In addition, two other restriction sites, EcoRI andHindIII, were also introduced at the 5′ ends of the N-terminal andC-terminal oligonucleotides, respectively, for convenient subcloning ofthe PCR-generated product. The approximately 1 kb rapAT14-encoding DNAwas amlplified by PCR using chromosomal DNA from Streptomyceshygroscopicus ATCC 29253 as template. The PCR conditions were asfollows: The 100 μL reaction mixture contains 10 μL of 10× ThermopolBuffer (New England Biolabs), 2% glycerol, 10% formamide, 100 pmoles ofeach oligo, 100-200 ng of template DNA and water to 84 μL. The samplewas then heated to 99° C. for two minutes followed by cooling to 80° C.for two minutes at which time 16 μL of a dNTP solution (1.25 mM dATP anddTTP, 1.5 dCTP and dGTP) and 1 μL of Vent_(R)® DNA Polymerase (NewEngland Biolabs) was added. Cycling was as follows: 30 cycles at 96.5°C./35 sec, 65° C./1 min and 72° C./1.5 min followed by one cycle at 72°C. for 3 min. The entire reaction was then run on a 1.2% low-meltingagarose gel and the desired fragment was isolated by melting theappropriate gel slice at 65° C., adding 3 volumes of TE buffer,extracting 2× with phenol and once with chloroform, and ethanolprecipitating the aqueous phase. The isolated DNA was ligated directlyinto HindI digested pUC19. The ligtation mixture was transformed into E.coli DH5α GIBCO BRL) according to the manufacturer's instructions andtransformants were selected on LB plates containing 150 μg/mL,ampicillin and 50 μL of a 2% solution of X-gal for bllue/whiteselectioni. Clones were confirmed by restriction analysis and thefidelity of the insert was confirmed by DNA sequencing. The finalplasmid construct was named pUC19/rapAT14.

EXAMPLE 14 Construction of plasmid pEryAT1/rapAT14

[0182] pEryAT1/rapAT14 was constructed using standard methods ofrecombinant DNA technology according to the schmatic outlines of FIG.24. To make a gene-replacement-vector specific for the eryAT1 domain,the two DNA regions immediately adjacent to eryAT1 were cloned andpositioned adjacent to the DNA encoding the rapAT14 domain in order toallow homologous recombination to occur. The strategy and protocol forconstructing the intermediate plasmid containing the flanking regions,pCS5/AT1-flank, are described in Example 3 and FIG. 9. To insert therapAT14 fragment between the flanking regions, pUC19/rapAT14 (fromExample 13) was digested with NsiI and AvrII and the resulting 1 kbfragment was isolated from a 0.8% agarose gel with Prep-A-Gene.pCS5/AT1-flank was also digested with these enzymes and the linerizedplasmid was isolated from 0.8% agarose gel. The two fragments wereligated, transformed into the intermediate host E. coli DH-5α andampicillin resistant clonies were selectec as previously described.Insertion of the rapAT14 fragment between the ery flanking regions wasconfirmed by restriction analysis and the resulting plasmid was calledpEryAT1/rapAT14.

EXAMPLE 15 Construction of Sfac. erythraea ER720 EryAT1/rapAT14

[0183] An example of a 12-desmethyl-12-deoxyerythromycin A producingmicroorganism was prepared by replacing the methymalonyl acyltransferasedomain of module 1 of the erythromycin PKS (EryAT1) of Sac. erythraeaER720 with the acyltransferase domain from module 14 of the rapamyycinPKS from S. hygroscopicus ATCC 29253. This was accomplished with therecombinant plasmid, pEryAT1/rapAT14, prepared as described in Example14. Transformation of Sac. erythraea ER720 and resolution of theintegration event were carried out according to the following method.Sac. erythraea ER720 cells were grown in 50 mL of SGGP medium (per 1liter aqueous solution: 4 g peptone, 4 g yeast extract, 4 g casaminoacids, 2 g glycine, 0.5 g MgSO₄.7 H₂O, 10 g glucose 20 mL of 500 mMKH₂PO₄) for 3 days at 32° C. and then washed in 10 mL of 10.3% sucrose.The cells were resuspended in 10 mL of P_(M) buffer containing 1 mg/mLlysozyme and incubated at 30° C. for 15-30 minutes until most of themycelial fragments were converted into spherical protoplasts. (P_(M)buffer per 1 liter aqueous solution: 200 g sucrose, 0.25 g K₂SO₄ in 890mL H₂O, with the addition after sterilization of 100 mL 0.25 M TES,pH7.2, 2 mL trace elements solution (Hopwood, et al, 1985, GeneticManipulation of Streptomyces A Laboratory Manual, The John InnesFoundation), 0.08 mL 2.5 M CaCl₂, 10 mL 0.5% KH₂PO₄, 2 mL 2.5M Mgcl₂.)

[0184] The protoplasts were washed once with P_(M) and then resuspendedin 3 mL of the same buffer.

[0185] Transformation was accomplished by centrifuging 200 μL ofprotoplasts for 15 seconds in a microfuge, decanting the supernatant,and resuspending the protoplasts in the P_(M) remaining in the tube.Tell μL of DNA solution was added (3 μL of pEryAT1/rapAT14 DNA fromExample 14 at about 1 μg/μL in 7 μL of P_(M) buffer) and mixed with theprotoplasts by gently tapping the tube. Two tenths of a milliliter of25% PEG 8000 in T buffer (Hopwood, et al, 1985, Genetic Manipulation ofStreptomyces A Laboratory Manual, The John Innes Institute) was thenadded, mixed by pipetting the solution 3 times and the suspensionimmediately spread on a dried R3M plate. The plate was incubated at 30°C. for 20 hours and overlaid with 2 mL of water containing 100 μg/mLthiostrepton, dried briefly and incubated 4 more days at 30° C.

[0186] To select stable transformants (integrants) colonies arising onthe transformation plates were re-streaked onto R3M plates containingthiostrepton (20 μg/mL). Four colonies were confirmed to be thiostreptonresistant and were inoculated into 30 mL of SGGP containing thiostrepton(10 μg/mL). After growth for 3 days, one mnL of each culture wasextracted with ethyl acetate as described in Example 5, and run on a TLCplate to confirm that the strains were no longer making erythromycin Adue to insertional inactivation by the integrating plasmid.

[0187] Integrants #1 and #4 were grown in SGGP without antibiotic forfour days and then plated onto non-selective R3M plates for sporulation.Spores were plated on R3M plates to obtain individual colonies, whichwere then screened for sensitivity to thiostrepton, indicating loss ofthe plasmid sequence from the chromosome. Six thiostrepton sensitivecolonies were isolated from integrant #4 and one of these (4-A-1) wasconfirmed by Southern hybrilizaition to have the EryAT1 replaced by therapAT14 (FIG. 25). Hybridization was at 65° C. and the stringency washwas with 0.1×SSC at 65° C. The strain was named Sac. erythraea ER720EryAT1/rapAT14.

EXAMPLE 16 Analysis of compounds produced by Sac. erythraea ER720EryAT1/rapAT14

[0188] Compounds produced by the recombinant Sac. erythraea strain,ER720 EryAT1/rapAT14, whose construction is described in Example 15,were characterized by TLC and mass spectrometry. For TLC analysis strain4-A-1 was grown in SCM medium (20 g Soytone, 15 g Soluble Starch, 10.5 gMOPS, 1.5 g Yeast Extract and 0.1 g CaCl₂ per liter of distilled H₂O)for 4 days at 30° C. The culture (1.5 mL) was centrifuged for 1 minutein a microfuge to remove cells. One mL of the resulting supernatant wasremoved to another microfuge tube and the pH adjusted to 9 by theaddition of 6 μL of NH₄OH. Then 0.5 mL of ethyl actcatte was added, thetube was vortexed for 10 sec and then centrifuged for approximately 5min to achieve phase separation. The organic phase was removed toanother tube, and the aqueous phase was re-extracted with 0.5 ml ofethyl acetate. The second organic phase was combined with the first anddried in a Speed Vac. The residue was taken up in 13 μL of ethyl acetateand 10 μL was spotted onto Merck 60F-254 silica gel TLC plate. The platewas run in isopropyl ether:metlanol:NH₄OH (75:35:2). Erythromycinderivatives were visualized by spraying the plates withanisaldehyde:sulfuric acid:ethanol (1:1:9). Using this reagent, a novelcompound predicted to be 12-desmethyl-12-deoxyerythromycin A, appearedas a blue spot running slightly faster than erythromycin A (FIG. 26).

[0189] To determine whether the novel spot seen on TLC has the molecularmass corresponding to the predicted 12-desmethyl-12-deoxyerythromycin A,an ethyl acetate extract was further analyzed by Mass Spectrometry. Sac.erythraea ER720 EryAT1/rapAT14 was grown in SCM medium for 4 days. TenmL of culture was centrifuged to remove mycelia and pH of thesupernatant was adjusted to 9 with NH₄OH. The supernatant was thenextracted twice with ethyl acetate and the organic phases pooled anddried. As shown in FIG. 33, mass spectrometric analysis of this crudeethyl acetate extract shows the mass of the novel spot to be 704, whichcorresponds to the molecular ion plus a proton (M+H^(+) of)12-desmethyl-12-deoxyerythromycin A.

EXAMPLE 17 Construction of plasmid pEryAT2/rapAT14

[0190] pEryAT2/rapAT14 was constructed using standard methods ofrecombinant DNA technology according to the schlematic outlines of FIGS.15 and 34. To make a gene-replacement-vector specific for the ery AT2domain, the two DNA regions immediately adjacent to ery AT2 were clonedand positioned adjacent to the DNA encoding the rapAT14 domain in orderto allow homologous recombination to occur. The strategy and protocolfor constructing the intermediate plasmid containing the flankingregions, pCS5/AT2-flank, are described in Example 6 and FIG. 14. Thefinal step in the construction of pEryAT2/rapAT14 was to ligate the 1 kbrapAT14-encoding DNA fragment having AvrII and NsiI ends topCS5/AT2-flank (Example 6) cut with the same enzymes to give the genereplacement/integration plasmid pEryAT2/rapAT14 (FIG. 27). All ligationswere transformed into the intermediate host E. coli DH15α and clonesselected as previously described.

EXAMPLE 18 Construction of Sac. erythraea ER720 EryAT2/rapAT14

[0191] A 10-desmethylerythromycin A and10-desmethyl-12-deoxyerythromycin A producing microorganism was preparedby replacing the DNA fragment encoding the methylmalonyl acyltransferasedomain of module 2 of the erythromycin PKS (EryAT2) of Sac. erythraeaER720 with a DNA fragment encoding a malonyl acyltransferase domain(rapAT14) from S. hygroscopicus ATCC 29253. This was accomplished withthe recomibinant plasmid, pEryAT2/rapAT14, pepared as described inExample 17. Transformation of ER720 and resolution of the integrationevent were carried out as described in Example 4 using 10 μL of DNAsolution consisting of 3 μL of pEryAT2/rapAT14 DNA at about 1 μg/μL in 7μL of P_(M) buffer. One thiostrepton resistant colony was isolated andwas inoculated into SGGP containing thiostrepton (10 μg/ml) to isolatechromosonal DNA for Southern analysis. Integration of the plasmid DNAinto the ER720 chromosome was further confirmed by Southernhybridization (data not shown). Hybridization was at 65° C. and thestringency wash was with 0.1×SSC at 65° C.

[0192] The confirmed integrant was grown in SGGP without antibiotic forfour days and then diluted 1000 fold into fresh medium and grown for 4more days. Protoplasts were then prepared and plated onto non-selectiveR3M plates to obtain individual colonies, which were then screened forsensitivity to thiostrepton, indicating loss of the plasmid sequencefrom the chromosome. Four thiostrepton sensitive colonies were selectedand 3 of these were confirmed by Southern hybridization, usingconditions described above, to have the EryAT2 replaced by rapAT14 (FIG.28). The strain was named Sac. erythraea ER720 EryAT2/rapAT14.

EXAMPLE 19 Analysis of compounds produced by Sac. erythraea ER70EryAT2/rapAT14

[0193] Compounds produced by the recombinant Sac. erythraea strain,ER720 EryAT2/rapAT14, whose construction is described in Example 18,were characterized by TLC, bioassay, and mass spectrometry.

[0194] For TLC analysis cells were grown in either SGGP or SCM medium(20 g Soytone, 15 g Soluble Starch, 10.5 g MOPS, 1.5 g Yeast Extract and0.1I g CaCl₂ per liter of distilled H₂O) for 4-5 days at 30° C. Culture(22 mL) was centrifuged for 5 minute to remove cells. The resultingsupernatant was removed to another tube and the pH adjusted to 9.0 bythe addition of 6 μL/mL of NH₄OH. Then an equal volume of ethyl acetatewas added, the liquid was mixed for 2 min. and then centrifuged forapproximately 5 min. to achieve phase separation. The organic phase wasremoved to another tube, and the aqueous phase was re-extracted withhalf volume of ethyl acetate. The second organic phase was combined withthe first and dried in a Speed Vac. The residue was taken up in 100 μLof ethyl atcetate and one fourth of the sample was spotted onto a Merck60F-254 silica gel TLC plate. The plate was run in isopropylether:nethanol:NH₄OH (75:35:2). Erythromycin derivatives were visualizedby spraying the plates with anlisaldehyde:sulfuric acid:ethanol (1:1:9).Using this reagent, two novel compounds predicted to be10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A,appeared as blue spots with the lower spot runninig slightly slower thanerythromycin A and upper spot running slightly faster than erythromycinA (FIG. 29).

[0195] To detect biological activity, a bioassay was performed. In thisassay, another fourth of the extracted sample from above was spottedonto a disc. The disc was then air-dried and placed over a platecontaining 50 mL of antibiotic medium II (DIFCO-BACTO) containingStaphylococcus aureus as an indicator strain. The inhibition zones weredeveloped by overnight incubation of the plate at 37° C. As shown inFIG. 30, the novel compounds have bioactivity.

[0196] To determine whether the novel spots seen on TLC have themolecular mass corresponding to the predicted 10-desmethylerythromycin Aand 10-desmethyl-12-deoxyerythromycin A, an ethyl acetate extract fromanother culture was further analyzed by mass spectrometry. The samplewas a crude extract of a 20 ml culture grown for 4 days. Massspectrometric analysis revealed the two novel compounds to have massesof 720 and 704, which correspond to the molecular ion plus a proton(M+H⁺) of 10-desmethylerythromycin A and10-desmethyl-12-deoxyerythromycin A, respectively.

EXAMPLE 20 Cloning of the ethylAT Domain from Streptomyces caelestis

[0197] A genomic library of Streptomyces caelestis NRRL-2821 U.S. PatNo. 3,218,239 issued Nov. 16, 1965) DNA was constructed in thebifunctional cosmid pNJ1 (Tuan, et al., Gene, 90: 21-29 (1990)). Cosmidvector was prepared by digesting 5 μg of pNJ1 with EcoRI,dephosphorylating with CIAP and then digesting with BglII to generateone arm and also digesting 5 μg of pNJ1 with HindIII, dephosphorylatingwith CIAP and then digesting with BglllI to generate the other. InsertDNA was prepared by partially digesting approximately 5 μg ofchromosomal S. caelestis NRRL-2821 DNA with SauIIIA according to theprocedure outlined in Maniatis et al., supra. Digestion conditions werechosen which produced fragment sizes of approximately 40 kb. Theligation was performed by mixing approximately 1 μg of the digestedchromosomal DNA with 0.5 μg of each cosmid arm. The ligation wasincubated at 16° C. overnight. Gigapackl XL (Stratagene®) was used forpackaging 2 μL of the ligation mix according the manufacturer'sinstructions. Transformation was done in E. coli L1-Blue MR cells(Stratagene®). Individiuial colonies were picked into thirty 96-wellplates to give a 99.99% probability that the library represented all S.caelestis NRRL-2821 genomic sequences.

[0198] The library was screened using a probe specific for the S.caelestis NRRL-2821 PKS region. The probe was generated by PCRamplification of S. caelestis NRRL-2821 genomic DNA using degenerateprimers designed from consensus ketosynthase (KS) and acyltransferase(AT) sequences in the GenBank database. The KS specific oligo (SEQ ID)NO:19) and the AT specific oligo (SEQ ID NO:20) generated a 900 bp PCRfragment. The PCR reaction contained 10 μL ThermolPol Buffer, 2 μLformamide, 25 μL of 20% glycerol, 3 μL 50 mM MgCl₂, 45 μL water, 50pmole of each primer, and approximately 0.2 μg DNA. The sample washeated to 99° C. for 5 minutes, and then placed on ice, at which time a10 μL cocktail consisting of 2 μL of a 10 mM mixture of dATP, dCTP,dGTP, and dTTP, 2 units of Vent DNA polymerase, and 7 μL of water wasadded. The sample was then transferred to a GeneAmp 9600 thermoocycler(Perkin Elmer, Foster City, Calif.) and a temperature cycle of 1 minuteat 95° C., 4 minutes at 5° C., and 4 minutes at 72° C. was repeated 30times, followed by a 15 minute incubation at 72° C. The desired PCRfragment was then isolated from 1.0% low melting agarose by standardprocedures. The KS/AT probe was made by labeling approximately 50 ng ofthe PCR fragment with ³²P using the Megaprime DNA Labeling System(Amersham Life Science, Arlington heights, Ill.). Library clones (2,880)were transferred from the 96-well plates to Hybond-N nylon filters(Amersham) and screened with the KS/AT probe according to procedures inManiaitis, et al., supra. Hybridization was performed at 65° C. and thefinal wash was in 0.1×SSC at 65° C. Nineteen of the clones hybridizedstrongly with the probe. These clones were then digested with SstI, runon a 1.0% agarose gel and transferred to Hybond-N nylon filters forSouthern analysis using the KS/AT probe (FIG. 31). The cosmid identifiedas pCEL18h5 was chosen for further analysis since it contained thelargest number of hybridizing restriction fragments.

[0199] The SstI fragments from cosmid pCEL18h5 were cloned into pGEM-3Zf(Promega, Madison, Wis.) and sequenced using the fmole DNA CycleSequencing System (Promega). The reactions were run on a Sequi-Gen IISequencing Apparatus (Bio-Rad, Hercules, Calif.). Individual fragmentswere oriented relative to one another by sequencing off of cosimidpCEL18h5 using primers that hybridized to the 5′ and 3′ ends of thefragments to genenate upstream and downstream sequence. These sequenceswere then matched with sequences from the individual fragments to placethem in the proper order. A very large SstI fragment (>10kb) was furtherdigested with SmaI to generate smaller fragments for cloning andsequencing.

[0200] By searching the GenBank database with the sequences obtained itwas possible to identify the various enzymatic motifs associated withthe niddamycin PKS cluster and to group these motifs into modules (seeFIG. 32) based on previous knowledge of Type I PKS organization. The C-6position of the niddamycin macrolactone ring has an aleldyde derivedfrom an ethyl side chain (FIG. 33). It was thus predicted that the AT ofmodule 5 of the niddamycin cluster is responsible for incorporating thisethyl group into the growing chain. In addition, the carbon at C-7 ofthe molecule is completely saturated leading to the prediction that ERand DH motifs would also be present in module 5. These motifs were, infact, found at the predicted region of the sequence. Furthermore, motifsfor the preceding 4 modules were as predicted, with an inactiveketoreductase motif in module 4 which leaves a keto group at C-9 of thering. Sequencing of that KR showed that the nucleotide binding siteGXGXXG (SEQ ID NO:27) was mutated to DXTXXP (SEQ ID NO:28). Thenucleotide sequence (SEQ ID NO:29) and corresponding amino acid sequence(SEQ ID NO:33) of the ethyl AT of module 5 are shown in FIG. 34 (top andbottom strands respectively).

[0201] A knockout experiment was also performd on this cluster,demonstrating that this sequence of DNA encodes the pathway forniddamycini biosynthesis (data not shown).

EXAMPLE 21 Construction of plasmid pEATE4

[0202] A multistep strategy was used to construct the plasmidpUC/ethAT/C6 (IFIG. 35), which consists of the DNA encoding the NidAT5domain flanked by approximately 2.0 kb of sequence upstream anddownstream from the eryAT4 encoding sequences, all contained in pUC19).EryAT4 flanking DNA was subcloned from pAIBX85. This plasmid is a pCS5derivative containing 8.4 kb of Sac. erythraea DNA from an XhoI site toa BamHI site in the eryAII gene of the erythromycin PKS cluster. Thesesites correspond to bases 23211 and 31581 , respectively, of GenBankaccession number M63676. The EryAT4 5-flanking DNA was isolated bydigesting pAIBX85 with MscI and BstEII (corresponding to nucleotides23,211 and 31,581, respectively). The resulting 1800 bp DNA fragment wastreated with the Klenow Fragment of DNA Polymerase I, ligated into theSmaI site of pUC 19, and transformed into E. coli DHF5α. Clones wereselected on LB plates containing 150 μg/mL ampicillin and 50 μL of a 2%solution of X-gal for blue/white selection. The clones were confirmed byrestriction analysis, resulting in the interinediate vectorpUC/5′-flank. For convenient cloning of the NidAT5-encoding sequences,an AvrII site was engineered at the 3′ end of the 5′ flanking DNA. Thiswas accomplished by PCR amplification from the PmlI site of the 5′flanking DNA to the BstEII site with two oligonucleotides (SEQ ID NO:21and SEQ ID NO:22). SEQ ID NO:22 incorporates an AvrII site and a BamHIsite at the 3′ end of the 5′ flanking DNA. PCR conditions were asdescribed in Example 20 using Sac. erythraea DNA as template with thefollowing changes: Taq polymerase (GIBCO BRL) was used with theaccompanying 10× buffer instead of Vent_(R)® DNA polymerase and cyclingconditions were 96° C./30 sec, 55° C./30 sec, 72° C./30 sec for 25cycles. The resulting 300 bp PCR fragment was then digested with PmlIand BamHI, gel purified from a 1.0% agarose gel with Prep-A-Gene, andligated back into pUC/5′ -flank digested with PmlI and BamHI to givepUC/5′-flanl-AvrII. The ligation was transformed into DH5α and platedonto LB plates containing 150 μg/mL ampicillin. Clones were confirmed byrestriction analysis and DNA sequencing.

[0203] In order to clone the NidAT5-encoding DNA fragment downstream ofthe 5′ flanking DNA, an AvrII site was also engineered at the 5′ end ofthe NidAT5-encoding DNA. As depicted in FIG. 36, an AvrII site could beengineered into the NidAT5 DNA without altering the amino acid sequence.Two PCR oligonucleotides (SEQ ID NO:23 and SEQ ID NO:24) were designedto create an AvrII site at the 5′ end and a BamHI site at the 3′ end,respectively, of the NidAT5-encoding DNA. A convenient FseI site occursnaturally at the 3′ end of NidAT5-encoding sequence, so the resultingPCR fragment contains an FseI site just upstream of the PCR engineeredBamHI site. SEQ ID NO:23 and SEQ ID NO:24 were used in at PCR reactionwith the template p16-2.2. This plasmid is pUC19 containing at 2.2 kbSmaI fragment from module 5 of the niddamycin PKS cluster (see FIG. 32),which encompasses) the sequences encoding NidAT5. The resulting 1.0 kbPCR fragment was digested with AvrIl and BamHI, purified from a1.0agarose gel using Prep-A-Gene, and cloned into the AvrIIl/BarmHI sitesof pUC/5′-flank-AvrIl. Clones were confirmed by restriction analysis andDNA sequencing, creating the intermediate plasmid pUC/5′- flank/ethAT.

[0204] The EryAT3′-flanking DNA was subcloned by digesting pAIBX85 withPmlI and MscI, corresponding to nucleotides 29,231 and 31,209,respectively, from the eryAII gene (GenBank accession number M63676).The DNA was gel purified on a 1.0% agarose gel using Prep-A-Gene andligated into the SmaI site of pUC19. The ligation was transformed intoDH5α and plated as described previously. Clones were confirmed byrestriction analysis, resulting in the plasmid pUC/3′-flank.

[0205] Attachment of the EryAT4 3′-flanking DNA to the NidAT5 -encodingsequence was accomplished by digesting plasmid pUC/3′-flank with FseIand BamHI, gel purifying the fragment from a 1.0% agarose gel usingPrep-A-Gene, and ligating it into pUC/5′-flank/ethAT that had beenpreviously digested with FseI and BamHI . The ligation was transformedinto DH5α as before and clones were analyzed by restriction analysis,resulting in the intermediate plasmid pUC/ethAT/C-6. The final step wasto remove the NidAT5/flanking DNA insert from pUC/ethAT/C-6 with EcoRIand HindIII and ligiate it into the EcoRI/HindIII sites of pCS5,resulting in the gene replacement integration plasmid pEAT4 (FIG. 37).

EXAMPLE 22 Construction of Sac. erythraea ER720 EAT4-46

[0206] An example of a 6-desmethyl-6-ethylerythromycin A producingmicroorganism was prepared by replacing the DNA fragment encoding themethymalonyl acyltransferase domain in module 4 of the erythromycin PKS(EryAT4) of Sac. erythraea ER720 with a newly discovered DNA fragmentencoding an ethylmalonyl acyltransferase domain (NidAT5) from S.caelestis NRRL-2821. This was accomplished using the recombinant plasmidpEAT4, prepared as described in Example 21. Transformation of Sac.erythraea ER720 and resolution of the integration event were carried outaccording to the procedures described in Example 4 using 10 μL of a DNAsolution consisting of 3 μL of pEAT4 DNA from Example 21 at about 1 μLin 7 μL of P_(M) buffer. One colony was confirmed to be thiostreptonresistant and was inoculated into SGGP containing thiostrepton (10μg/mL) to isolate chromosomal DNA for Southern analysis. Integration ofthe plasmid DNA into Sac. erythraea ER720 was confirmed by Southernanalysis (data not shown). Hybridization was at 65° C. and thestringency wash was with 0.1×SSC at 65° C.

[0207] The confirmed integrant was then subcultured into 30 ml SGGPwithout antibiotic using 10 μL of the previous culture. After three daysgrowth at 30° C. the strain was again subcultured into 30 mL of freshSGGP as before and plated onto nonselective R3M plates for sporulation.Spores were plated on R3M plates to obtain individual colonies, whichwere then screened for sensitivity to thiostrepton, indicating loss ofthe plasmid sequence from the chromosome. Nine thiostrepton sensitivecolonies were isolated and three of them were confirmed by Southernhybridization to have the EryAT4 replaced by NidAT5 (FlG. 38).Hybridization was at 65° C. and the stringency wash was with 0.0×SSC at65° C. The strain was named Sac. erythraea ER720 EAT4-46, referred to assimply EAT4-46.

EXAMPLE 23 Analysis of compounds produced by EAT4-46

[0208] Compounds produced by strain EAT4-46, whose construction isdescribed in Example 22, were characterized by TLC, bioautography andmass spectrometry.

[0209] The cells were grown in 30 mL of SCM for 4-5 days at 3° C. Theculture was centrifuged for 10 minutes in a Sorval GLC-4 GeneralLaboratory Centrifuge at setting 10 to remove cells. Thie resultingsupernatant was adjusted to pH 9.0 by the addition of 180 μL of NH₄OH.Then 15 ml of ethyl acetate was added, the tube was vortexed for 30seconds and then centrifuged for 10 minutes to achieve phase separation.The organic phase was removed to another tube, and the aqueous phase wasre-extracted with 15 ml of ethyl acetate. The second organic phase wascombined with the first and dried in a Speed-Vac. The residue was takenup in 30 μL of ethyl acetate and 10 μL was spotted onto a Merck 60F-254silica gel TLC plate. The plate was run in a solvent containingisolpropyl ether:methanol:NH₄OH (75:35:2). Erythromycin derivatives werevisualized by spraying the plates with anisaldehyde:sulfuricacid:ethanol (1:1:9). The results showed that EAT4-46 produced acompound that migrated with the same r_(f) as erythromycin A produced bywild type Sac. erytjraea ER720, except in much lower yield (data notshown).

[0210] To determine the molecular mass of the compound, all ethylacetate extract was prepared from a 50 mL SCM culture of EAT4-46 asdescribed above, using a proportionate amount of reagents. The resultingresidue was taken up in 50 μL of ethyl acetate and run on a TLC plate asdescribed previously, except that the plate was not sprayed withanisaldehyude. THE compound of interest was isolated by scraping thesilicai resin in the vicinity of the spot and extracting the resin asdescribed in Example 8. Mass spectrometric analysis revealed that thecompound produced by the EAT4-46 strain had a mass of 734, whichcorresponds to the molecular ion plus a proton (M+M⁺) of erythromycin A.

[0211] In an attempt to increase substrate pools for the NidAT5ethylmalonyl AT construction, the EAT4-46 strain was grown in 100 mL ofSCM media containing 50 mM butyric acid, pH 7.0. The culture was grownfor 4 days at 3° C. and then centrifuged for 10 minutes in a SorvalGLC-4 Centrifuge to pellet the cells. The resulting supernatant wasadjusted to pH 9.0 by the addition of 600 μL of NH₄OH and extractedtwice with ½ volumes of ethyl acetate as described previously. Afterdrying in a Speed-Vatc rotary concentrator, the extracted material wastaken up in 100 μl of ethyl acetate and 10 μl was used for TLC analysisas described previously. Two spots running near eryA were observed inthe butyric acid fed culture as opposed to only one sport in SCM mediaalone (FIG. 39). To determine the molecular mass of the two spots, mostof the remainder of the extract was again subjected to TLC, and thecompounds in the eryA region of the plate were isolated is describedpreviously. Mass spectrometric analysis revealed that the two spots hadmolecular masses of 734 and 748. A molecular mass of 734 corresponds tothe molecular ion plus a proton (M+H⁺) of erythromycin A, whereas thespecies of molecular mass 748 is consistent with the molecular mass plusa proton (M+H⁺) of ethylerythromycin A.

EXAMPLE 24 Cloning of the NidAT6 Domain from Streptomyces caelestisNRRL-2821

[0212] A genomic library of Streptomyces caelestis NRRL-2921 DNA wasgenerated and screened with a probe specific for PKS genes as describedin Example 20. From Southern analysis of SstI digests of the positiveclones (FIG. 31), some clones were selected for further analysis. Theseclones were digested with SmaI and run on a 1% agarose gel for Southernhybridization with the PKS specific probe. The analysis revealed that asecond cosmid, pCEL13f5, shared many hybridizing bandes with pCEL18h5,but also contained two unique bands of 1.9 kb and 6.0 kb (FIG. 40). Thiscosmid was chosen for further analysis in order to determine thesequence of the remaining PKS genes in the niddamycin pathway. CosmidpCEL13f5 was digested with SstI and the fragments were ligated tp pUC19.A large SstI fragment (>10 kb) was further digested with SmaI andligated to pUC19. The ligations were transformed into DH5α cells andclones were selected on LB plates containing 150 μg/mL ampicillin and 50μl of a 2% solution of X-gal for blue/white selection. DNA from clonescontaining the appropriate insert was isolated using the QlAprep SpinPlasmid Kit (QIAGEN Inc., Chatsworth, Calif.). Subclones were selecedusing the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit(Perkin Elmer), and the reactions were run on a 4.75% acrylamide, 8.3 Murea gel in an Applied Biosystems 373 DNA Sequencing System. Ordering ofthe inserts and motif identification was done as described in Example20.

[0213] The insert in cosmid pCEL13f5 was found to be approximately 25 kbin length, and the 5′ end of the insert had about 10 kb of identicalsequence with the 3′ end of the insert in pCEL18h5. Together, the twocosmids contain all of the PKS genes of the niddamycin pathway (FIG.32). Based on the structure of niddamycin (FIG. 33), the AT contained inmodule 6 (NidAT6) may utilize hydroxymalonate (tartronate) in thebiosynthesis of the C-3, C-4, and O-4 positions of the macrolactone ringof niddamycin. (S. Omura et al. (J. Anibiotics 36:611-613 (1983)) havesuggested that glycolate may be incorporated in the biosynthesis of theC-3, C-4 and O-4 positions of leucomycin, a closely related 16-memberedmacrolide). The nucleotide sequence of NidAT6 (top strantd, SEQ IDNO:30) and its corresponding amino acid sequence (lower strand, SEQ IDNO:34) are shown in FIG. 41. A comparison of the amino acid sequence ofNidAT6 with other ATs in the Swissprot database shows that NidAT6resembles methylalonyl ATs (data not shown).

EXAMPLE 25 Construction of plasmid pUC18/NidAT6

[0214] Two PCR oligonucleotides (SEQ ID NO:25 and SEQ ID NO:26) aredesigned to subclone the 1024 bp DNA fragment encoding the NidAT6 domainfrom the niddamycin PKS cluster and to introduce two unique restrictionsites, AvrII and NsilI, for cassette cloning. This necessitatesnucleotide changes, show in bold in FIG. 42, at the beginning and nearthe end of the NidAT6-encoding DNA sequence. The changes shown alsocause the replacement of a proline codon near the N-terminus of theNidAT6 domain with a valine codon, in order to increase the similarityof the domain junction sequence to that found naturally for some of theAT domains of the rapamycin PKS. (In FIG. 42, the underlined nucleotidesare the wild-type sequence.) In addition, two other restriction sites,EcoRI and BglII, are also introduced at the 5′ ends of the N-terminaland C-terminal oligonucleotides, respectively, for convenient subcloningof the PCR-generated product. The approximately 1 kb NidAT6 domainencoding DNA is amplified using methods described in Reagents andGeneral Methods from Cosmid pCEL13f5. The PCR product is digested withEcoRI and BglII and subcloned into the EcoRI and BamHI sites of pUC18.The ligation mixture is transformed into E. coli DH5α (GIBCO BRL)according to the manufacturer's instructions and transformants areselected on LB plates containing 150 ,μg/mL ampicillin and 50 μL of a 2%solution of X-gal for blue/white selection. Clones are confirmed byrestriction analysis and the fidelity of the insert is confirmed by DNAsequencing. The final plasmid construct is named pUC18/NidAT6.

EEXAMPLE 26 Construction of plasmid pEryAT2/NidAT6

[0215] pEryAT2/NidAT6 is constructed using standard methods ofrecombinant DNA technology according to the schematic outlines of FIGS.15 and 53. To make a gene-replacement-vector specific for the eryAT2domain, the two DNA regions immediately adjacent to eryAT2 are clonedand positioned adjacent to the DNA encoding the NidAT6 domain in orderto allow homologous recombination to occur. The strategy and protocolfor constructing the intermediate plasmid containing the flankingregions, pCS5/AT2-flank, are described in Example 6 and FIG. 13. Thefinal step in the construction of pEryAT2/NidAT6 is to ligate the 1 kbNidAT6-encoding DNA fragment having AvrII and NsiI ends topCS5/AT2-flank (Example 6) cut with the same enzymes to give the genereplacement/integration plasmid pEryAT2/NidAT6 (FIG. 43). All ligationmixes are transformed into the intermediate host E. coli DH5α and clonesare selected and charatcterized as described previously.

EXAMPLE 27 Construction of Sac. erthraea ER720 EryAT2/NidAT6

[0216] A 10-desmethyl-10-hydroxyerythromycin A and12-deoxy-10-desmetlyl-10-hydroxyerythromycin A producing microorganismis prepared by replacing the DNA fragment encoding the methylmalonylacyltransferase domain of module 2 of the erythromycin PKS (EryAT2) ofSac. erythraea ER720 with a DNA fragment encoding a hydroxymalonylacyltransferase domain (NidAT6) from S. caelestis NRRL-2821. This isaccomplished with the recombinant plasmid, pEryAT2/NidAT6, prepared asdescribed in Example 26. Transformation of ER720 and resolution of theintegration event are carried out as described in Example 4 using 10 μLof DNA solution consisting of 3 μL of pEryAT2/NidAT6 DNA at about 1 g/μLin 7 μL of P_(M) buffer. Thiostrepton resistant colonies are isolatedand inoculated into SGGP containing thiostrepton (10 μg/mL) to isolatechromosomal DNA for Southern analysis. Integration of the plasmnid DNAinto the ER720 chromosome is further confirmed by Southernhybridization. Hybridization is at 65° C. and the stringency wash iswith 0.1×SSC at 65° C.

[0217] Confirmed integrants are grown in SGGP without antibiotic forfour days and then diluted 1000-fold into fresh medium and grown for 4more days. Protoplasts are then prepared and plated onto non-selectiveR3M plates to obtain individual colonies, which are screened forsensitivity to thiostrepton, indicating loss of the plasmid sequencefrom the chromosome. Thiostrepton sensitive colonies are then selectedand these are confirmed by Southern hybridization, using condition;described above, the EryAT2 replaced by NidAT6. The strain is designatedSac. erythraea ER720EryAT2/NidAT6.

EXAMPLE 28 Analysis of compounds prodticed by Sac. erythraea ER720EryAT2/NidAT6

[0218] Compounds produced by the recombinant Sac. erythraea strain,ER720 EryAT2/NidAT6, whose construction is described in Example 27, arecharacterized by TLC, bioassay, and mass spectrometry.

[0219] For TLC analysis cells are grown in either SGGP or SCM medium (20g Soytone, 15 g Soluble Starch, 10.5 g MOPS, 1.5 g Yeast Extract and0..1 g CaCl₂ per liter of distilled H₂O) for 4-5 days at 30° C. Theculture is centrifuged for 5 min. to remove cells. The resultingsupernatant is removed to another tube and the pH adjusted to 9.0 by theaddition of 6 μL/mL of NH₄OH. Then an equal volume of ethyl acetate isadded, the liquid is mixed for 2 min. and then centrifuged forapproximately 5 min. to achieve phase separation. The organic phase isremoved to another tube, and the aqueous phase is re-extracted with ahalf volume of ethyl acetate. The second organic phase is combined withthe first and dried in a Speed Vac. The residue is taken up inapproximately 25 μL of ethyl acetate and 15 μL are spotted onto a Merck60F-254 silicai gel TLC plate. The plate is run in isopropylether:methanol:NH₄OH (75:35:2). Erythromycin derivatives are visualizedby spraying the plates with anisaldehyde:sulfuric acid:ethanol (1:1:9).Using this reagent, two novel compounds predicted to be10-desmethyl-10-hydoxyerythromycin A and12-deoxy-10-desmethyl-10-hydroxyerythromycin A, are expected to appearas blue spots running slightly slower than erythromycin A.

[0220] To determine whether the novel spots seen on TLC have themolecular mass corresponding to the predicted10-desmethyl-10-hydroxyerythromycini A and12-deoxy-10-desmethyl-10-hydroxyerythromycin A, the remaining extract isfurther analyzed by mass spectrometry. The two novel compounds arepredicted to have masses of 736 and 720, which correspond to themolecular ion plus a proton (M+H⁺) of10-desmethyl-10-hydroxyerythromycin A and12-deoxy-10-desmethyl-10-hydroxyerythromycin A, respectively.

1 34 925 base pairs nucleic acid double linear 1 GGGCCGCTGG CGGTGATGTTCACCGGACAG GGCTCCCAAC GCCCCGGCAT GGGACGACAG 60 TTGTACGAGC ACTTCCCCGTCTTCGCCCAG GCACTGGACG AGGTCTTCGC ACTCGCCACC 120 CCCGGACTAC GCGAGGTGATGTTCGACCCC GACCAGGCCG AAACACTCCA ACGCACCGAC 180 CACGCCCAGA TCGCCCTGTTCGCCTTCGAA ACCGCCCTCT ACCGACTCTG GGAATCCTGG 240 GGCCTGCGAC CCGACATGGTCTGCGGACAC TCGGTCGGAG AAATCACCGC AGCCCACGTC 300 TCCGGCACCC TCACCCTCCCCGACGCCGTC CACCTCGTCA CCACACGCGG CACCCTCATG 360 CAAAACCTGC CCCCCGGCGGCGCCATGCTC GCCGTCGCCA CCGACCCCCA CACCCTCCAA 420 CCCCACCTCG ACAACCACCACGACACCATC TCCATCGCCG CCATCAACGG CCCCCACGCC 480 ACCGTCCTCT CCGGCGACCGCACCACCCTC CACCACATCG CCACCCAACT CAACACCAAA 540 CCCTTCACCA CCACCCTCAACACCCTCACC CACCACCCCC CACACACACC CCTCATCAGC 600 ATGCTCACCG CCACACCCACCCACCCCGAC ACCACCCACT GGACCCAGCA CATCACCGCA 660 CCCGTCCGCT ACACCGACACCCTCCACCAC CTCCACCACC ACGGCATCAC CACCTACCTC 720 GAAATCGGCC CCGACACCACCCTCACCGCC CTCGCCCGCA CCACCCTCCC CACCACCACC 780 CACCTCATCC CCACCACCCGCCGCAACCAC AACGAAGTCC GCAGCACGAA CGAGGCGTTG 840 GGCAGGGTGT TCAGCGTGGGCCACTCGGTG GACTGGCGGG CCCTCACTCC GACCGGGAGG 900 CGTACCTCCC TGCCGACGTACCCCT 925 1030 base pairs nucleic acid double linear 2 CCTAGGACGGCAGTCCTGCT CACCGGGCAG GGTTCCCAGC GTCAGGGCAT GGGGCGCGAA 60 CTGTACGACCGGTCACCGGT GTTCGCCGCC TCGTTCGACG CGATCTGCGC TCAACTCGAC 120 GGGCAACTGCCTCGTCCCCT CAAGGACGTT CTCTTCGCCC CCGAGGGGTC GGAGGACGCC 180 GCGCTCATCGACCGTACGGT GTTCACACAG GCGGCTCTGT TCGCCGTGGA GACCTCCCTG 240 TTCCGGCTGTTCGAGGCCCA CGGCCTCGTC CCCGACTACC TCATCGGCCA CTCCATCGGC 300 GAAGTGACCGCGGCCCACCT GGCCGGGGTC CTCGATCTGG CGGACGCGTG CGTCCTGGTC 360 GCCCACCGCGGCCGCCTGAT GCAGTCGGCC CGGGCCGGCG GCGCGATGGC CGCGGTCCAG 420 GCGAGCGAGGACGAGGTACG CGAGGCCCTC GCGACCTTCG ACGATGCGGT TGCCGTGGCC 480 GGAGTCAACGGCCCGAACGC CACCGTCGTC TCCGGCGACG AGGACGCGGT CGAGCGGCTG 540 GTCGCGCGCTGGCGCGAGCA GGGCAGGCGG ACGAAGCGGC TGCCGGTCAG CCACGCCTTC 600 CACTCGCCGCACATGGACGG GATCGTCGAC GAGTTCGTCA CCGCCGTCTC CGGGCTCACC 660 TTCCGCTCCCCGACGATCCC GGTCGTCTCC AACGTCACCG GGACCCTCGC CACCGTCGAC 720 CAGCTGACCTCGCCCGCGTA CTGGGCACGC CACATCCGCG AGGCCGTGCG CTTCGCCGAC 780 GGGGTGCGGTACCTGGAGGG CGAGGGCGTC ACCGAATGGC TGGAGCTCGG GCCCGACGGC 840 GTTCTCGTCGCCCTGGTCGA GGACTGCCTG GCGAAGGAGG CGGGATCGCT CGCGTCCGCC 900 CTGCGCAAGGGGGCGAGCGA GCCCCACACC GTGGGCGCGG CCATGGCCCG CGCGGTGCTG 960 CGCGGATCCGGCCCCGACTG GGCGGCGGTG TTCCCCGGCG CACGGCGGGT CGACCTTCCG 1020 ACGTATGCAT1030 35 base pairs nucleic acid single linear 3 ATCTACACST CSGGCACSACSGGCAAGCCS AAGGG 35 35 base pairs nucleic acid single linear 4CTSAAGGCSG GCGGCGCSTA CGTSCCSATC GACCC 35 30 base pairs nucleic acidsingle linear 5 CGCGAATTCC TAGGCTGGCG GTGATGTTCA 30 33 base pairsnucleic acid single linear 6 GCCGGATCCA TGCATACGTC GGCAGGGAGG TAC 33 28base pairs nucleic acid single linear 7 GCTCGAATTC GCTGGTCGCG GTGCACCT28 32 base pairs nucleic acid single linear 8 GACGGATCCG GCCCTAGGCTGCGCCCGGCT CG 32 30 base pairs nucleic acid single linear 9 TTGGGATCCTATGCATTCCA GCGCGAGCGC 30 26 base pairs nucleic acid single linear 10GAGAAGCTTG GCGCGACTTG CCCGCT 26 36 base pairs nucleic acid single linear11 TTTTTTAAGC TTGGTACCTG CTCACCGGCA ACACCG 36 42 base pairs nucleic acidsingle linear 12 TTTTTTGGAT CCCTGCAGCC TAGGGTCGGA GGCACTGCCG GT 42 37base pairs nucleic acid single linear 13 TTTTTTCTGC AGTATGCATTCCAGGGCAAG CGGTTCT 37 36 base pairs nucleic acid single linear 14TTTTTTGAAT TCACGCGTTG CCCGCGGCGT AGGCGC 36 34 base pairs nucleic acidsingle linear 15 GATCGAATTC CCTAGGACGG CAGTCCTGCT CACC 34 35 base pairsnucleic acid single linear 16 GATCGGATCC ATGCATACGT CGGAAGGTCG ACCCG 3536 base pairs nucleic acid single linear 17 TTCGAAGAAT TCCCTAGGGTTGCCTTCCTG TTCGAC 36 36 base pairs nucleic acid single linear 18TTCGAAAAGC TTATGCATAG ACCGGCAGAT CCACCG 36 19 base pairs nucleic acidsingle linear 19 CGGTSAAGTC SAACATCGG 19 20 base pairs nucleic acidsingle linear 20 GCRATCTCRC CCTGCGARTG 20 44 base pairs nucleic acidsingle linear 21 GAGAGAGGAA CCAACGCGCA CGTGATCGTC GAAGAGGCAC CAGC 44 45base pairs nucleic acid single linear 22 GAGAGAGGAT CCGACCTAGGCGCGGAGGTC ACCGGCGCGA CGGCG 45 43 base pairs nucleic acid single linear23 GAGAGACCTA GGAAGCCGGT GTTCGTGTTC CCCGGCCAGG GCT 43 47 base pairsnucleic acid single linear 24 GAGAGAGGAT CCGAGGCCGG CCGTGCGCCCGGACCGAAGA CCGCCTC 47 41 base pairs nucleic acid single linear 25GAGAGAATTC CCTAGGGTCG CCTTCGTCTT TCCCGGGCAG G 41 37 base pairs nucleicacid single linear 26 TTGAGATCTT ATGCATACGA GGGAAGCGGC ACCCTGC 37 37base pairs nucleic acid single linear 27 TTGAGATCTT ATGCATACGAGGGAAGCGGC ACCCTGC 37 37 base pairs nucleic acid single linear 28TTGAGATCTT ATGCATACGA GGGAAGCGGC ACCCTGC 37 1010 base pairs nucleic aciddouble linear 29 GCCGACCGTG TCGTGTTCGT GTTCCCCGGC CAGGGCTCGC AGTGGGCCGGAATGGCCGAG 60 GGGCTGCTGG AGCGGTCCGG CGCGTTCCGG AGTGCGGCCG ACTCGTGCGACGCCGCGCTG 120 CGGCCGTACC TCGGCTGGTC GGTGCTGAGC GTGCTGCGCG GGGAACCGGACGCGCCCTCG 180 CTCGACCGGG TCGACGTCGT GCAGCCGGTG CTGTTCACGA TGATGGTCTCGCTCGCGGCG 240 GTCTGGCGTG CGCTGGGGGT GGAACCGGCG GCGGTCGTCG GGCACTCGCAGGGTGAGATC 300 GCCGCTGCCC ATGTCGCCGG TGCGCTGTCG CTGGACGACT CGGCCCGGATCGTCGCCCTG 360 CGCAGTCGGG CGTGGCTCGG ACTGGCGGGC AAGGGCGGCA TGGTGGCGGTGCCGATGCCG 420 GCGGAGGAGC TGCGGCCGCG GCTGGTGACG TGGGGGGACC GTCTGGCCGTCGCCGCCGTC 480 AACAGCCCCG GTTCCTGCGC CGTCGCAGGC GACCCGGAGG CGCTGGCCGAACTGGTGGCG 540 CTGCTGACCG GTGAGGGGGT GCACGCCCGG CCGATCCCCG GCGTCGACACGGCGGGCCAC 600 TCGCCGCAGG TGGACGCGTT GCGGGCTCAT CTGCTGGAGG TGCTGGCCCCGGTCGCCCCC 660 CGACCGGCCG ACATCCCGTT CTACTCGACG GTGACCGGCG GGCTGCTGGACGGCACCGAG 720 CTGGACGCGA CGTACTGGTA CCGCAACATG CGCGAGCCCG TCGAGTTCGAGCGGGCCACA 780 CGGGCGCTGA TCGCCGACGG GCACGACGTC TTCCTGGAGA CGAGCCCGCATCCCATGCTG 840 GCCGTGGCGC TGGAGCAGAC GGTCACCGAC GCCGGCACCG ACGCGGCGGTGCTCGGGACC 900 CTGCGCCGCC GCCACGGCGG TCCTCGCGCG CTGGCCCTGG CCGTCTGCCGCGCCTTCGCG 960 AGGCGGTCTT CGGTCCGGGC GCACGGCCCG TGGAGTTGCC CACCTATCCG1010 1035 base pairs nucleic acid double linear 30 CGCGCGCCTG CCTTCGTCTTTCCCGGGCAG GGCGCCCAGT GGGCCGGACT GGGAGCGCGG 60 CTCCTCGCGG ACTCCCCCGTCTTCCGCGCC AGGGCCGAGG CATGCGCGCG GGCGCTGGAG 120 CCTCACCTCG ACTGGTCGGTCCTCGACGTG CTGGCCGGCG CCCCGGGCAC CCCTCCCATC 180 GACCGGGCCG ACGTGGTGCAGCCGGTGCTG TTCACCACGA TGGTCTCGCT GGCCGCCCTC 240 TGGGAGGCCC ACGGGGTGCGGCCGGCCGCG GTCGTGGGCC ACTCCCAGGG CGAGGTGGCC 300 GCGGCCTGCG TGGCCGGTGCCCTGTCGCTG GACGACGCTG CCCTGGTGAT CGCCGGACGC 360 AGCAGGCTGT GGGGGCGGCTGGCCGGGAAC GGCGGGATGC TCGCGGTGAT GGCTCCGGCC 420 GAGCGGATCC GTGAGCTGCTCGAACCATGG CGGCAGCGGA TTTCGGTGGC GGCGGTCAAT 480 GGCCCCGCCT CGGTCACCGTCTCCGGTGAC GCGCTCGCGC TGGAGGAGTT CGGCGCGCGG 540 CTCTCCGCCG AGGGGGTGCTGCGCTGGCCG CTGCCGGGCG TCGACTTCGC CGGCCACTCG 600 CCGCAGGTGG AGGAGTTCCGCGCTGAGCTC CTGGACCTGC TCTCCGGCGT ACGGCCGGCT 660 CCTTCGCGGA TACCTTTCTTCTCCACCGTG ACGGCGGGTC CTTGCGGCGG CGACCAGCTG 720 GACGGGGCGT ACTGGTACCGCAACACGCGC GAACCCGTGG AGTTCGACGC CACGGTCCGG 780 GCGCTGCTGC GTGCGGGCCATCACACGTTC ATCGAGGTCG GTCCGCATCC GCTGCTCAAC 840 GCCGCGATCG ACGAGATCGCAGCGGACGAG GGGGTAGCGG CCACGGCCCT GCATACGCTC 900 CAGCGGGGCG CTGGCGGCCTTGACCGCGTG CGCAACGCGG TGGGCGCCGC TTTCGCGCAC 960 GGTGTCCGGG TCGACTGGAACGCCCTGTTC GAGGGCACCG GTGCGCGCAG GGTGCCGCTT 1020 CCCTCGTACG CCTTC 1035328 amino acids amino acid single linear None 31 Gly Pro Leu Ala Val MetPhe Thr Gly Gln Gly Ser Gln Arg Pro Gly 1 5 10 15 Met Gly Arg Gln LeuTyr Glu His Phe Pro Val Phe Ala Gln Ala Leu 20 25 30 Asp Glu Val Phe AlaLeu Ala Thr Pro Gly Leu Arg Glu Val Met Phe 35 40 45 Asp Pro Asp Gln AlaGlu Thr Leu Gln Arg Thr Asp His Ala Gln Ile 50 55 60 Ala Leu Phe Ala PheGlu Thr Ala Leu Tyr Arg Leu Trp Glu Ser Trp 65 70 75 80 Gly Leu Arg ProAsp Met Val Cys Gly His Ser Val Gly Glu Ile Thr 85 90 95 Ala Ala His ValSer Gly Thr Leu Thr Leu Pro Asp Ala Val His Leu 100 105 110 Val Thr ThrArg Gly Thr Leu Met Gln Asn Leu Pro Pro Gly Gly Ala 115 120 125 Met LeuAla Val Ala Thr Asp Pro His Thr Leu Gln Pro His Leu Asp 130 135 140 AsnHis His Asp Thr Ile Ser Ile Ala Ala Ile Asn Gly Pro His Ala 145 150 155160 Thr Val Leu Ser Gly Asp Arg Thr Thr Leu His His Ile Ala Thr Gln 165170 175 Leu Asn Thr Lys Thr Asn Trp Leu Asn Val Ser His Ala Phe His Ser180 185 190 Pro Leu Met Gln Pro Ile Leu Gln Pro Phe Thr Thr Thr Leu AsnThr 195 200 205 Leu Thr His His Pro Pro His Thr Pro Leu Ile Ser Met LeuThr Ala 210 215 220 Thr Pro Thr His Pro Asp Thr Thr His Trp Thr Gln HisIle Thr Ala 225 230 235 240 Pro Val Arg Tyr Thr Asp Thr Leu His His LeuHis His His Gly Ile 245 250 255 Thr Thr Tyr Leu Glu Ile Gly Pro Asp ThrThr Leu Thr Ala Leu Ala 260 265 270 Arg Thr Thr Leu Pro Thr Thr Thr HisLeu Ile Pro Thr Thr Arg Arg 275 280 285 Asn His Asn Glu Val Arg Ser ThrAsn Glu Ala Leu Gly Arg Val Phe 290 295 300 Ser Val Gly His Ser Val AspTrp Arg Ala Leu Thr Pro Thr Gly Arg 305 310 315 320 Arg Thr Ser Leu ProThr Tyr Pro 325 343 amino acids amino acid single linear None 32 Pro ArgThr Ala Val Leu Leu Thr Gly Gln Gly Ser Gln Arg Gln Gly 1 5 10 15 MetGly Arg Glu Leu Tyr Asp Arg Ser Pro Val Phe Ala Ala Ser Phe 20 25 30 AspAla Ile Cys Ala Gln Leu Asp Gly Gln Leu Pro Arg Pro Leu Lys 35 40 45 AspVal Leu Phe Ala Pro Glu Gly Ser Glu Asp Ala Ala Leu Ile Asp 50 55 60 ArgThr Val Phe Thr Gln Ala Ala Leu Phe Ala Val Glu Thr Ser Leu 65 70 75 80Phe Arg Leu Phe Glu Ala His Gly Leu Val Pro Asp Tyr Leu Ile Gly 85 90 95His Ser Ile Gly Glu Val Thr Ala Ala His Leu Ala Gly Val Leu Asp 100 105110 Leu Ala Asp Ala Cys Val Leu Val Ala His Arg Gly Arg Leu Met Gln 115120 125 Ser Ala Arg Ala Gly Gly Ala Met Ala Ala Val Gln Ala Ser Glu Asp130 135 140 Glu Val Arg Glu Ala Leu Ala Thr Phe Asp Asp Ala Val Ala ValAla 145 150 155 160 Gly Val Asn Gly Pro Asn Ala Thr Val Val Ser Gly AspGlu Asp Ala 165 170 175 Val Glu Arg Leu Val Ala Arg Trp Arg Glu Gln GlyArg Arg Thr Lys 180 185 190 Arg Leu Pro Val Ser His Ala Phe His Ser ProHis Met Asp Gly Ile 195 200 205 Val Asp Glu Phe Val Thr Ala Val Ser GlyLeu Thr Phe Arg Ser Pro 210 215 220 Thr Ile Pro Val Val Ser Asn Val ThrGly Thr Leu Ala Thr Val Asp 225 230 235 240 Gln Leu Thr Ser Pro Ala TyrTrp Ala Arg His Ile Arg Glu Ala Val 245 250 255 Arg Phe Ala Asp Gly ValArg Tyr Leu Glu Gly Glu Gly Val Thr Glu 260 265 270 Trp Leu Glu Leu GlyPro Asp Gly Val Leu Val Ala Leu Val Glu Asp 275 280 285 Cys Leu Ala LysGlu Ala Gly Ser Leu Ala Ser Ala Leu Arg Lys Gly 290 295 300 Ala Ser GluPro His Thr Val Gly Ala Ala Met Ala Arg Ala Val Leu 305 310 315 320 ArgGly Ser Gly Pro Asp Trp Ala Ala Val Phe Pro Gly Ala Arg Arg 325 330 335Val Asp Leu Pro Thr Tyr Ala 340 344 amino acids amino acid single linearNone 33 Ala Asp Arg Val Val Phe Val Phe Pro Gly Gln Gly Ser Gln Trp Ala1 5 10 15 Gly Met Ala Glu Gly Leu Leu Glu Arg Ser Gly Ala Phe Arg SerAla 20 25 30 Ala Asp Ser Cys Asp Ala Ala Leu Arg Pro Tyr Leu Gly Trp SerVal 35 40 45 Leu Ser Val Leu Arg Gly Glu Pro Asp Ala Pro Ser Leu Asp ArgVal 50 55 60 Asp Val Val Gln Pro Val Leu Phe Thr Met Met Val Ser Leu AlaAla 65 70 75 80 Val Trp Arg Ala Leu Gly Val Glu Pro Ala Ala Val Val GlyHis Ser 85 90 95 Gln Gly Glu Ile Ala Ala Ala His Val Ala Gly Ala Leu SerLeu Asp 100 105 110 Asp Ser Ala Arg Ile Val Ala Leu Arg Ser Arg Ala TrpLeu Gly Leu 115 120 125 Ala Gly Lys Gly Gly Met Val Ala Val Pro Met ProAla Glu Glu Leu 130 135 140 Arg Pro Arg Leu Val Thr Trp Gly Asp Arg LeuAla Val Ala Ala Val 145 150 155 160 Asn Ser Pro Gly Ser Cys Ala Val AlaGly Asp Pro Glu Ala Leu Ala 165 170 175 Glu Leu Val Ala Leu Leu Thr GlyGlu Gly Val His Ala Arg Pro Ile 180 185 190 Pro Gly Val Asp Thr Ala GlyHis Ser Pro Gln Val Asp Ala Leu Arg 195 200 205 Ala His Leu Leu Glu ValLeu Ala Pro Val Ala Pro Arg Pro Ala Asp 210 215 220 Ile Pro Phe Tyr SerThr Val Thr Gly Gly Leu Leu Asp Gly Thr Glu 225 230 235 240 Leu Asp AlaThr Tyr Trp Tyr Arg Asn Met Arg Glu Pro Val Glu Phe 245 250 255 Glu ArgAla Thr Arg Ala Leu Ile Ala Asp Gly His Asp Val Phe Leu 260 265 270 GluThr Ser Pro His Pro Met Leu Ala Val Ala Leu Glu Gln Thr Val 275 280 285Thr Asp Ala Gly Thr Asp Ala Ala Val Leu Gly Thr Leu Arg Arg Arg 290 295300 His Gly Gly Pro Arg Ala Leu Ala Leu Ala Val Cys Arg Ala Phe Ala 305310 315 320 His Gly Val Glu Val Asp Pro Glu Ala Val Phe Gly Pro Gly AlaArg 325 330 335 Pro Val Glu Leu Pro Thr Tyr Pro 340 345 amino acidsamino acid single linear None 34 Arg Ala Pro Ala Phe Val Phe Pro Gly GlnGly Ala Gln Trp Ala Gly 1 5 10 15 Leu Gly Ala Arg Leu Leu Ala Asp SerPro Val Phe Arg Ala Arg Ala 20 25 30 Glu Ala Cys Ala Arg Ala Leu Glu ProHis Leu Asp Trp Ser Val Leu 35 40 45 Asp Val Leu Ala Gly Ala Pro Gly ThrPro Pro Ile Asp Arg Ala Asp 50 55 60 Val Val Gln Pro Val Leu Phe Thr ThrMet Val Ser Leu Ala Ala Leu 65 70 75 80 Trp Glu Ala His Gly Val Arg ProAla Ala Val Val Gly His Ser Gln 85 90 95 Gly Glu Val Ala Ala Ala Cys ValAla Gly Ala Leu Ser Leu Asp Asp 100 105 110 Ala Ala Leu Val Ile Ala GlyArg Ser Arg Leu Trp Gly Arg Leu Ala 115 120 125 Gly Asn Gly Gly Met LeuAla Val Met Ala Pro Ala Glu Arg Ile Arg 130 135 140 Glu Leu Leu Glu ProTrp Arg Gln Arg Ile Ser Val Ala Ala Val Asn 145 150 155 160 Gly Pro AlaSer Val Thr Val Ser Gly Asp Ala Leu Ala Leu Glu Glu 165 170 175 Phe GlyAla Arg Leu Ser Ala Glu Gly Val Leu Arg Trp Pro Leu Pro 180 185 190 GlyVal Asp Phe Ala Gly His Ser Pro Gln Val Glu Glu Phe Arg Ala 195 200 205Glu Leu Leu Asp Leu Leu Ser Gly Val Arg Pro Ala Pro Ser Arg Ile 210 215220 Pro Phe Phe Ser Thr Val Thr Ala Gly Pro Cys Gly Gly Asp Gln Leu 225230 235 240 Asp Gly Ala Tyr Trp Tyr Arg Asn Thr Arg Glu Pro Val Glu PheAsp 245 250 255 Ala Thr Val Arg Ala Leu Leu Arg Ala Gly His His Thr PheIle Glu 260 265 270 Val Gly Pro His Pro Leu Leu Asn Ala Ala Ile Asp GluIle Ala Ala 275 280 285 Asp Glu Gly Val Ala Ala Thr Ala Leu His Thr LeuGln Arg Gly Ala 290 295 300 Gly Gly Leu Asp Arg Val Arg Asn Ala Val GlyAla Ala Phe Ala His 305 310 315 320 Gly Val Arg Val Asp Trp Asn Ala LeuPhe Glu Gly Thr Gly Ala Arg 325 330 335 Arg Val Pro Leu Pro Ser Tyr AlaPhe 340 345

What is claimed is:
 1. A compound of the formula:

wherein R₁, R_(2,) R_(3,) R_(4,) R_(5,) and R₆ are independentlyselected from Q wherein Q is selected from the group consisting of (a)—H, (b) -Me, (c) -Et, and (d) —OH; L₁ and L₂ are independently —H or—OH; L₃ is D-desosamine or —OH; and L₄ is L-mycarose, L-cladinose or —OHwith the proviso that when R₁-R₅ are -Me, R₆ is other than —H or -Me. 2.The compound of claim 1 wherein Q is selected from the group consistingof (a), (b), and (c) and L₁, L_(2,) L₃ and L₄ are as defined therein. 3.The compound of claim 1 wherein Q is selected from the group consistingof (a), (b), and (d) and L_(1,) L_(2,) L₃ and L₄ are as defined therein.4. The compound of claim 1 wherein Q is selected from the groupconsisting of (a), (c), and (d) and L_(1,) L_(2,) L₃ and L₄ are asdefined therein.
 5. The compound of claim 1 wherein Q is selected fromthe group consisting of (b), (c), and (d) and L_(1,) L_(2,) L₃ and L₄are as defined therein.
 6. The compound of claim 1 wherein Q is selectedfrom the group consisting of (a) and (b) and L_(1,) L_(2,) L₃ and L₄ areas defined therein.
 7. The compound of claim 1 wherein Q is selectedfrom the group consisting of (a) and (c) and L_(1,) L_(2,) L₃ and L₄ areas defined therein.
 8. The compound of claim 1 wherein Q is selectedfrom the group consisting of (a) and (d) and L_(1,) L_(2,) L₃ and L₄ areas defined therein.
 9. The compound of claim 1 wherein Q is selectedfrom the group consisting of (b) and (c) and L_(1,) L_(2,) L₃ and L₄ areas defined therein.
 10. The compound of claim 1 wherein Q is selectedfrom the group consisting of (b) and (d) and L_(1,) L_(2,) L₃ and L₄ areas defined therein.
 11. The compound of claim 1 wherein Q is selectedfrom the group consisting of (c) and (d) and L_(1,) L_(2,) L₃ and L₄ areas defined therein.
 12. The compound of claim 1 wherein Q is (a) and L₁,L_(2,) L₃ and L₄ are is defined therein.
 13. The compound of claim 1wherein Q is (c) and L₁, L_(2,) L₃ and L₄ are as defined therein. 14.The compound of claim 1 wherein Q is (d) and L₁, L_(2,) L₃ and L₄ are asdefined therein.
 15. The compound of claim 1 wherein (a) R₆ and R₁ are—H and R₂, R₃, R₄ and R₅ are -Me, (b) R₅ and R₁ are —H and R₂, R₃, R₄and R₆ are -Me, (c) R₄ and R₁ are —H and R₂, R₃, R₅ and R₆ are -Me, (d)R₃ and R₁ are —H and R₂, R₄, R₅ and R₆ are -Me, (e) R₂ and R₁ are —H andR₃, R₄, R₅ and R₆ are -Me, (f) R₆ and R₂ are —H and R₁, R_(3 l , R) ₄and R₅ are -Me, (g) R₅ and R₂ are —H and R₁, R₃, R₄ and R₆ are -Me, (h)R₄ and R₂ are —H and R₁, R₃, R₅ and R₆ are -Me, (i) R₃ and R₂ are —H andR₁, R₄, R₅ and R₆ are -Me, (j) R₆ and R₃ are —H and R₁, R₂, R₄ and R₅are -Me, (k) R₅ and R₃ are —H and R₁, R₂, R₄ and R₆ are -Me, (l) R₄ andR₃ are —H and R₁, R₂, R₅ and R₆ are -Me, (m) R₆ and R₄ are —H and R ₁,R₂, R₃ and R₅ are -Me, (n) R₅ and R₄ are —H and R ₁, R₂, R₃ and R₆ are-Me, (o) R₆ and R₅ are —H and R ₂, R₃ and R₄ are -Me, and L₁, L₂, L₃ andL₄ are as defined therein.
 16. The compound of claim 15 wherein (a)-(o)are as defined therein, L₁ and L₂ are —OH, L₃ is D-desosamine and L₄ isL-cladinose.
 17. The compound of claim 1 wherein (a) R₆, R₂ and R₁ are—H and R₃, R₄ and R₅ are -Me, (b) R₅, R₂ and R₁ are —H and R₃, R₄ and R₆are -Me, (c) R₄, R₂ and R₁ are —H and R₃, R₅ and R₆ are -Me, (d) R₃, R₂and R₁ are —H and R₄, R₅ and R₆ are -Me, (e) R₆, R₃ and R₁ are —H andR₂, R₄ and R₅ are -Me, (f) R₅, R₃ and R₁ are —H and R₂, R₄ and R₆ are-Me, (g) R₄, R₃ and R₁ are —H and R₂, R₅ and R₆ are -Me, (h) R₆, R₄ andR₁ are —H and R₂, R₃ and R₅ are -Me, (i) R₅, R₄ and R₁ are —H and R₂, R₃and R₆ are -Me, (j) R₆, R₅ and R₁ are —H and R₂, R₃ and R₄ are -Me, (k)R₆, R₃ and R₂ are —H and R₁, R₄ and R₅ are -Me, (l) R₅, R₃ and R₂ are —Hand R₁, R₄ and R₆ are -Me, (m) R₄, R₃ and R₂ are —H and R₁, R₅ and R₆are -Me, (n) R₆, R₄ and R₂ are —H and R₁, R₃ and R₅ are -Me, (o) R₅, R₄and R₂ are —H and R₁, R₃ and R₆ are -Me, (p) R₆, R₅ and R₂ are —H andR₁, R₃ and R₆ are -Me, (q) R₆, R₄ and R₃ are —H and R₁, R₂ and R₅ are-Me, (r) R₅, R₄ and R₃ are —H and R₁, R₂ and R₆ are -Me, (s) R₆, R₅ andR₃ are —H and R₁, R₂ and R₄ are -Me, or (t) R₆, R₅ and R₄ are —H and R₁,R₂ and R₃ are -Me, and L₁, L₂, L₃ and L₄ are as defined therein.
 18. Theconmpound of claim 17 wherein (a)-(t) are as defined therein, L₁ and L₂are —OH, L₃ is D-desosamine and L₄ is L-cladinose.
 19. The compound ofclaim 1 wherein (a) R₆, R₃, R₂ and R₁ are —H and R₅, and R₄ are -Me, (b)R₅, R₃, R₂ and R₁ are —H and R₆, and R₄ are -Me, (c) R₄, R₃, R₂ and R₁are —H and R₅, and R₆ are -Me, (d) R₆, R₄, R₂ and R₁ are —H and R₃, andR₅ are -Me, (e) R₅, R₄, R₂ and R₁ are —H and R₃, and R₆ are -Me, (f) R₆,R₅. R₂ and R₁ are —H and R₃, and R₄ are -Me, (g) R₆, R₄, R₃ and R₁ are—H and R₂, and R₅ are -Me, (h) R₅, R₄, R₃ and R₁ are —H and R₂, and R₆are -Me, (i) R₆, R₅, R₄ and R₁ are —H and R₂, and R₃ are -Me, (j) R₂,R₄, R₃ and R₂ are —H and R₁, and R₆ are -Me, (k) R₆, R₄, R₃ and R₂ are—H and R₁, and R₆ are -Me, (l) R₆, R₄, R₃ and R₂ are —H and R₁, and R₆are -Me, (m) R₆, R₅, R₃ and R₂ are —H and R₁, and R₄ are -Me, or (n) R₆,R₅, R₄ and R₃ are —H and R₁, and R₂ are -Me, and L₁, L₂, L₃ and L₄ areas defined therein.
 20. The compound of claim 19 wherein (a)-(n) are asdefined therein, L₁ and L₂ are —OH, L₃ is D-desosamine and L₄ isL-cladinose.
 21. The compound of claim 1 wherein (a) R₅, R₄, R₃, R₂ andR₁ are —H and R₆ is -Me, (b) R₆, R₄, R₃, R₂ and R₁ are —H and R₅ is -Me,(c) R₆, R₅, R₃, R₂ and R₁ are —H and R₄ is -Me, (d) R₆, R₅, R₄, R₂ andR₁ are —H and R₃ is -Me, (e) R₆, R₅, R₄, R₃ and R₂ are —H and R₂ is -Me,or (f) R₆, R₅, R₄, R₃ and R₂ are —H and R₁ is -Me, and L₁, L₂, L₃ and L₄are as defined therein.
 22. The compound of claim 21 wherein (a)-(f) areas defined therein, L₁ and L₂ are —OH, L₃ is D-desosamine and L₄ isL-cladinose.
 23. The compound of claim 1 wherein R₁, R₂, R₃, R₄, R₅ andR₆ are —H and L₁, L₂, L₃ and L₄ are as defined therein.
 24. The compoundof claim 23 wherein R_(1,) R_(2,) R₃, R₄, R₅ and R₆ are as definedtherein, L₁ and L₂ are —OH, L₃ is D-desosamine and L₄ is L-cladinose.25. The compound of claim 1 selected from the group consisting of6,10-didesmethlyl-6-ethylerythromycin A;10,12-didesmethyl-12-deoxy-12-ethylerythromycin A;10,12-didesmethyl-12-deoxy-10-hydroxyerythromycin A;6,10,12-tridesmethyl-6,12-diethylerythromycin A, and6,10,12-tridesmethyl-6-deoxy-6,12-diethylerythlromycin A.
 26. Thecompound of claim 1 selected from the group consisting of10-desmethylerythronolide B, 10-desmethyl-6-deoxyerythronolide B,12-desmethylerythronolide B, 12-desmethyl-6-deoxyerythronolide B,12-desmethyl-12-ethylerythronolide B,6-desmethyl-6-deoxy-6-ethylerythronolide B, 10-desmethylerythromycin A,10-desmethyl-12-deoxyerythromycin A,10-desmethyl-6,12-dideoxyerythromycin A, 12-desmethylerythromycin A,12-desmethyl- 12-deoxyerythromycin A,12-desmethyl-6,12-dideoxyerythromycin A,6-desmethyl-6-6-ethtlerthromycin A, 12-desmethyl-12-ethylerythromycin A,12-desmethyl-12-deoxy-12-ethylerythromycin A,10-desmethyl-10-hydroxyerythromycin A,12-desmethyl-12-epihydroxyerythromycin A, 10,12-didesmethylerythromycinA, 10,12-didesmethyl-12-deoxyerythromycin A, and10,12-didesmethyl-6,12-dideoxyerythromycin A.
 27. The compound of claim1 selected from the group consisting of 10-desmethylerthronolide B,10-desmethyl-6-deoxyerythronolide B, 12-desmethylerythronolide B,12-desmethyl-6-deoxyerythronolide B 10-desmethylerythromycin A,10-desmethyl-12-deoxyerythromycin A, 10-desmethyl-6,12-deoxyerythromycinA, 12-desmethyl-6,12-dideoxyerythromycin A,10,12-didesmethylerythromycin A, 10,12didesmethyl-12-deoxyerythromycinA, 10,12-didesmethyl-6,12-didteoxyerythromycin A.
 28. A compoundselected from the group consisting of 10-desmethylerythromycin A,10-desmethyl-12-deoxyerythromycin A, and12-desmethyl-12-deoxyerythromycin A.
 29. An isolated polynucleotidesequence or fragment thereof which encodes an enzymatically activeacyltransferase domain from a polyketide-producing microorganismselected from the group consisting of Streptomyces hygroscopicus,Streptomyces venezuelae, and Streptomyces caelestis.
 30. Thepolynucleotide of claim 29 selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:29 and SEQ ID NO:30.
 31. The polynucleotideof claim 29 wherein said acyltransferase domain is selected from thegroup consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ IDNO:34.
 32. A vector comprising a polynucleotide sequence or fragmentthereof which encodes an enzymatically active acyltransferase domainfrom Streptomyces.
 33. The vector of claim 32 wherein said Streptomycesis selected from the group consisting of Streptomyces hygroscopicus,Streptomyces venezuelae, and Streptomtces caelestis.
 34. The vector ofclaim 32 which is pCS5.
 35. The vector of claim 32 wherein saidpolynucleotide is selected from the group consisting of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:29 and SEQ ID NO:30.
 36. The vector of claim 32wherein said acyltransferase domain is selected from the groupconsisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.37. A vector selected from the group consisting of pUC18/LigAT2,pEryAT1/LigAT2, pEryAT2/LigAT2, pUC18/venAT, pEryAT1/venAT,pUC19/rapAT14, pEryAT1/rapAT14, pEryAT2/rapAT14, pUC/5′-flank/ethAT,pUC/ethAT/C-6, pEAT4, pUC18/NidAT6, and pEryAT2/NidAT6.
 38. A host celltransformed with the vector of claim
 32. 39. The host cell of claim 38wherein said cell is a bacterial cell.
 40. The host cell of claim 39wherein said bacterial cell is selected from the group consisting of E.coli and BacilIus species.
 41. The host cell of Claim 40 wherein saidcell is a polyketide-producing microorganism.
 42. The host cell of claim41 wherein said polyketide-producing microorganism is selected from thegroup consisting of Saccharopolyspora species, Nocardia species,Micromonospor species, Arthrobacter species, Streptomyces species,Actinomadura species, and Dactylosporangium species.
 43. The host cellof claim 42 wherein said polyketide-producing microorgianism is selectedfrom the group consisting of Saccharopolyspora hirsuta, Micromonosporarosaria, Micromonospora megalomicea, Streptomyces antibioticus,Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyceshygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis,Streptomyces fradiae, Streptomyces plantensis, Streptomycesviolaceoniger, Streptomyces ambofaciens, Streptomyces griseoplanus, andStreptomyces venezuelae.
 44. The host cell of claim 42 wherein saidpolyketide-producing microorganism is selected from the group consistingof Saccharopolyspora species and Streptomyces species.
 45. The host cellof claim 44 wherein said polyketide-producing microorganism isSaccharopolyspora erythraea.
 46. The host cell of claim 44 wherein saidpolyketide-producing microorganism is selected from the group consistingof Streptomyces hygroscopicus, Streptomyces venezuelae, and Streptomycescaelestis.
 47. A method for altering the substrate specificity of apolykeitde synthase in a first polyketide-producing microorganismcomprising the steps of : (a) isolating a first and second genomic DNAsegment, each comprising a polyketide synthase wherein said first geomicDNA segment is from said first polyketide-producing microorganism andsaid second genomic DNA segment is from said first polykeitde-producingmicroorganism or a second polyketide-producing microorganism; (b)identifying one of more descrete fragments of said first genomic DNAsegment, each of which encodes an acyltransferase domain: (c)identifying one or more discrete fragments of said second genemic DNAsegment, each of which encodes a related domain to said acyltranseferasedomain of said first genomic DNA segment; and (d) transforming a cell ofsaid first polyketide-producing microorganism with one or more of saidfragments from step (c) under conditions suitable for the occurrence ofa homologous recombination event, leading to the replacement of one ormore of said fragments from said first genomic DNA segment with one ormore of said fragments from step (c).
 48. The method of claim 47 whereinsaid first polyketide-producing microorganism is Saccharopolysporaerythraea.
 49. The method of claim 47 wherein said secondpolyketide-producing microorganism is Streptomyces.
 50. The method ofclaim 49 wherein said Streptomyces is selected from the group consistingof Streptomyces antibioticus, Streptomyces mycarofaciens, Streptomycesavermitilis, Streptomyces hygroscopicus, Streptomyces caelestis,Streptomyces tsukubaensis, Streptomyces fradiae, Streptomyces platensis,Streptomyces violaceoniger, Streptomyces ambofaciens, and Streptomycesvenezuelae.
 51. The method of claim 50 wherein said Streptomyces isselected from the group consisting of Streptomyces caelestis,Streptomyces hygroscopicus, and Streptomyces venezuelae.
 52. The methodof claim 47 wherein said first polyketide-producing microorganism isStreptomyces.
 53. The method of claim 52 wherein said Streptomyces isselected from the group consisting of Streptomyces antibioticus,Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyceshygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis,Streptomyces fradiae, Streptomyces platensis, Streptomycesviolaceoniger, Streptomyces ambofaciens, and Streptomyces venezuelae.54. The method of claim 53 wherein said Streptomyces is selected fromthe group consisting of Streptomyces caelestis, Streptomyceshygroscopicus, and Streptomyces venezuelae.
 55. The method of claim 47wherein said second polyketide-producing microorganism isSaccharopolyspora erythraea.
 56. The method of claim 47 wherein saidrelated domain is selected from the group consisting of SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.